IMMUNO-BASED BOTULINUM TOXIN SEROTYPE A ACTIVITY ASSAYS

Abstract

The present specification discloses SNAP-25 compositions, methods of making .alpha.-SNAP-25 antibodies that bind an epitope comprising a carboxyl-terminus at the P.sub.1 residue from the BoNT/A cleavage site scissile bond from a SNAP-25 cleavage product, .alpha.-SNAP-25 antibodies that bind an epitope comprising a carboxyl-terminus at the P.sub.1 residue from the BoNT/A cleavage site scissile bond from a SNAP-25 cleavage product, methods of detecting BoNT/A activity, and methods of detecting neutralizing .alpha.-BoNT/A antibodies.

Description

[0001] This application is a continuation of U.S. patent application Ser. No. 12/723,474, filed Mar. 12, 2010, which is a continuation-in-part of Ser. No. 12/403,531, filed Mar. 13, 2009, which claims priority pursuant to 35 U.S.C. .sctn. 119(e) to U.S. Provisional Patent Application Ser. No. 61/036,723 filed Mar. 14, 2008, all incorporated entirely herein by reference.

SEQUENCE LISTING

[0002] The application contains a Sequence Listing that has been filed electronically in the form of a text file, created Aug. 17, 2020, and named "18383-US-CIP1CON1-NTB-SubstituteSequenceListing.txt" (313573 bytes), the contents of which are incorporated herein by reference in their entirety.

[0003] The ability of Clostridial toxins, such as, e.g., Botulinum neurotoxins (BoNTs), BoNT/A, BoNT/B, BoNT/C1, BoNT/D, BoNT/E, BoNT/F and BoNT/G, and Tetanus neurotoxin (TeNT), to inhibit neuronal transmission are being exploited in a wide variety of therapeutic and cosmetic applications, see e.g., William J. Lipham, Cosmetic and Clinical Applications of Botulinum Toxin (Slack, Inc., 2004). Clostridial toxins commercially available as pharmaceutical compositions include, BoNT/A preparations, such as, e.g., BOTOX.RTM. (Allergan, Inc., Irvine, Calif.), DYSPORT.RTM./RELOXIN.RTM., (Ipsen Ltd., Slough, England), PURTOX.RTM. (Mentor Corp., Santa Barbara, Calif.), XEOMIN.RTM. (Merz Pharmaceuticals, GmbH., Frankfurt, Germany), NEURONOX.RTM. (Medy-Tox, Inc., Ochang-myeon, South Korea), BTX-A (Biogen-tech Ltd., University, Yantai, Shandong, China); and BoNT/B preparations, such as, e.g., MYOBLOC.RTM./NEUROBLOC.RTM. (Solstice Neurosciences, Inc., South San Francisco, Calif.). As an example, BOTOX.RTM. is currently approved in the U.S. for the treatment of cervical dystonia in adults to decrease the severity of abnormal head position and neck pain associated with cervical dystonia; for the treatment of severe primary axillary hyperhidrosis that is inadequately managed with topical agents; and for the treatment of strabismus and blepharospasm associated with dystonia, including benign essential blepharospasm or VII nerve disorders in patients 12 years of age and above.

[0004] At present the mouse LD.sub.50 bioassay, a lethality test, remains the "gold standard" used by all pharmaceutical manufacturers to express the potency of their preparations. S. S. Arnon et al., JAMA 285: 1059-1070 (2001). In fact, the units on the pharmaceutical preparations' labels are mouse LD.sub.50 units and the number of animals needed to produce statistically useful LD.sub.50 data is large. The advantage of the mouse LD.sub.50 bioassay is that it measures all the steps necessary for botulinum toxin uptake (e.g., toxin binding to a cell surface receptor, internalization of the toxin-receptor complex, light chain translocation into the cytoplasm, light chain cleavage of substrate), instead of merely determining the activity for only part of this intoxication process, such as, e.g., in vitro assays that only measure light chain enzymatic activity. Unfortunately, the mouse LD.sub.50 bioassay suffers from many drawbacks including high operational cost due to the large numbers of laboratory animals required, a lack of specificity since all BoNT serotypes will cause the same measurable end-point, and the potential for inaccuracy unless large animal groups are used. In addition, animal rights groups have exerted pressure on regulatory agencies in the U.S. (FDA/NICEATM/ICCVAM) and Europe (MHRA and EDQM), and on pharmaceutical companies manufacturing botulinum neurotoxin products to reduce animal testing and more importantly replace the mouse LD.sub.50 bioassay for product release. The regulatory agencies are engaging pharmaceutical companies to apply the three "Rs" principle to the potency testing of botulinum neurotoxins: Reduce, Refine, Replace. D. Straughan, Progress in Applying the Three Rs to the Potency Testing of Botulinum Toxin Type A, Altern. Lab. Anim. 34(3): 305-313 (2006). In recent years, several steps have been already taken to reduce and refine the mouse LD.sub.50 bioassay in order to standardize the protocol and produce more consistent data using fewer animals per assay.

[0005] Thus, a simple, reliable, validated and governmental agency acceptable botulinum toxin activity assay that can evaluate the integrity of all the steps necessary in botulinum toxin uptake would be of significant value because such a non-animal based assay would alleviate the need for animal testing and all the disadvantages, costs and ethical concerns associated with this type of animal-based assay. The present specification provides novel compositions, cells, and methods for assaying the activity of a botulinum toxin A useful for various industries, such as, e.g., the pharmaceutical and food industries, and provides related advantages as well. Such compositions, cells, and methods do not use live animals or tissues taken from live animals, but can evaluate all the steps necessary for neurotoxin action.

DETAILED DESCRIPTION OF THE DRAWINGS

[0006] FIGS. 1A-1B are schematics of the current paradigm of neurotransmitter release and Clostridial toxin intoxication in a central and peripheral neuron. FIG. 1A shows a schematic for the neurotransmitter release mechanism of a central and peripheral neuron. The release process can be described as comprising two steps: 1) vesicle docking, where the vesicle-bound SNARE protein of a vesicle containing neurotransmitter molecules associates with the membrane-bound SNARE proteins located at the plasma membrane; and 2) neurotransmitter release, where the vesicle fuses with the plasma membrane and the neurotransmitter molecules are exocytosed. FIG. 1B shows a schematic of the intoxication mechanism for tetanus and botulinum toxin activity in a central and peripheral neuron. This intoxication process can be described as comprising four steps: 1) receptor binding, where Clostridial toxin binds to a Clostridial receptor complex and initiates the intoxication process; 2) complex internalization, where after toxin binding, a vesicle containing a toxin/receptor system complex is endocytosed into the cell; 3) light chain translocation, where multiple events are thought to occur, including changes in the internal pH of the vesicle, formation of a channel pore comprising the H.sub.N domain of Clostridial toxin heavy chain, separation of the Clostridial toxin light chain from the heavy chain, and release of the light chain and 4) enzymatic target modification, where the light chain of Clostridial toxin proteolytically cleaves its target SNARE substrates, such as, e.g., SNAP-25, VAMP or Syntaxin, thereby preventing vesicle docking and neurotransmitter release.

[0007] FIGS. 2A-2B show a comparison of BoNT/A uptake in four cell lines by Western blot analysis. FIG. 2A shows a graph of SNAP-25 cleavage product detected based on amount of BoNT/A used to treat the cell line. The data were analyzed in SigmaPlot using a 4 parameter logistic model and EC.sub.50 values were obtained for each cell line. Ranking of SNAP-25 cleavage product signals detected was: SiMa>>Neuro-2a>LA1-55n>PC12. FIG. 2B shows the signal-to-noise ratios of the raw signals at 300 pM vs. 0 pM and 1.2 pM vs. 0 pM were calculated for the assay. SiMa cells generated the highest signal-to-noise ratios and the lowest EC.sub.50 values.

[0008] FIG. 3 shows optimization of cell differentiation media for established cell lines useful in an immuno-based method of detecting BoNT/A activity disclosed in the present specification.

[0009] FIG. 4 shows optimization of cell differentiation time for cells comprising an established cell line useful in an immuno-based method of detecting BoNT/A activity disclosed in the present specification.

[0010] FIG. 5 shows optimization of BoNT/A treatment of cells comprising an established cell line useful in an immuno-based method of detecting BoNT/A activity disclosed in the present specification. The results indicate an EC.sub.50 of less than 2 pM was achieved with any of the BoNT/A treatments tested.

[0011] FIG. 6 shows the sensitivity of an immuno-based method of detecting BoNT/A activity disclosed in the present specification. The results indicated that uptake of BoNT/A by the cells took less than one minute before producing significant amounts of SNAP-25 cleavage product over background.

[0012] FIG. 7 shows the specificity of an immuno-based method of detecting BoNT/A activity disclosed in the present specification. The results indicate that the immuno-based methods of detecting BoNT/A activity disclosed in the present specification can measure all the steps involved in BoNT/A intoxication.

[0013] FIG. 8 shows a dose response curve of differentiated SiMa cells treated with a BoNT/A complex using an immuno-based method of detecting BoNT/A activity disclosed in the present specification.

[0014] FIG. 9 shows the results of an immuno-based BoNT/A activity assay for a formulated BoNT/A pharmaceutical product using an immuno-based method of detecting BoNT/A activity disclosed in the present specification.

[0015] FIGS. 10A-10B show the detection of neutralizing .alpha.-BoNT/A antibodies in human serum using an immuno-based method of detecting BoNT/A activity disclosed in the present specification.

DETAILED DESCRIPTION

[0016] The present specification provides novel assays for determining the presence or absence of an active BoNT/A in a sample and for determining the activity/potency of a BoNT/A preparation. The novel cell-based assays disclosed in the present specification rely on cells, reagents and detection methods that enable the assay to detect picomolar quantities of BoNT/A in a sample. The cell-based assays disclosed in the present specification reduce the need for animal toxicity studies, yet serve to analyze multiple functions BoNT/A, namely, binding and cellular uptake of toxin, translocation into the cell cytosol, and protease activity. As discussed further below, the novel methods and compositions can be used to analyze crude and bulk samples as well as highly purified di-chain toxins and formulated toxin products and further are amenable to automated high throughput assay formats.

[0017] Thus, one aspect disclosed in the present specification provides compositions for producing .alpha.-SNAP-25 antibodies that can bind an epitope comprising a carboxyl-terminus at the P.sub.1 residue from the BoNT/A cleavage site scissile bond from a SNAP-25 cleavage product. Compositions can comprise an adjuvant and a composition including a SNAP-25 antigen, a carrier linked to a SNAP-25 antigen, or a carrier linked to a flexible spacer linked to a SNAP-25 antigen, where the flexible linker intervenes between the SNAP-25 antigen and the carrier. It is envisioned that any and all SNAP-25 antigens that triggers an immune response that produce a .alpha.-SNAP-25 antibody that can bind an epitope comprising a carboxyl-terminus at the P.sub.1 residue from the BoNT/A cleavage site scissile bond from a SNAP-25 cleavage product can be useful as a SNAP-25 antigen, including, without limitation, a SNAP-25 antigen derived from a naturally occurring SNAP-25, a SNAP-25 antigen derived from a non-naturally occurring SNAP-25, and a SNAP-25 antigen comprising an immunoreactive fragment of the SNAP-25, the SNAP-25 from a naturally occurring SNAP-25 or a non-naturally occurring SNAP-25. SNAP-25 antigens useful for producing .alpha.-SNAP-25 antibodies that can bind an epitope comprising a carboxyl-terminus at the P.sub.1 residue from the BoNT/A cleavage site scissile bond from a SNAP-25 cleavage product include, without limitation, SNAP-25 antigens comprising a SNAP-25 peptide having a carboxylated C-terminal glutamine linked to a carrier peptide, including, without limitation SEQ ID NO: 38. Other compositions useful for making .alpha.-SNAP-25 antibodies that can bind an epitope comprising a carboxyl-terminus at the P.sub.1 residue from the BoNT/A cleavage site scissile bond from a SNAP-25 cleavage product include, without limitation, a composition comprising a carrier linked to a flexible linker linked to a SNAP-25 antigen a carboxylated C-terminal glutamine, wherein the flexible linker intervenes between the SNAP-25 antigen and the carrier. It is envisioned that any and all adjuvants can be useful in such a composition, including, without limitation, polyethylene glycol (PEG), monomethoxypolyethylene glycol (mPEG), polyvinyl alcohol (PVA), complete and incomplete Freund's adjuvant.

[0018] Another aspect disclosed in the present specification provides methods of producing an .alpha.-SNAP-25 antibody that can bind an epitope comprising a carboxyl-terminus at the P.sub.1 residue from the BoNT/A cleavage site scissile bond from a SNAP-25 cleavage product. Aspects of this method comprise the steps of (a) administering to an animal a composition disclosed in the present specification; (b) collecting from the animal a sample containing an .alpha.-SNAP-25 antibody or .alpha.-SNAP-25 antibody-producing cell; and (c) isolating the .alpha.-SNAP-25 antibody from the sample. The methods disclosed are useful for making either .alpha.-SNAP-25 monoclonal antibodies that can bind an epitope comprising a carboxyl-terminus glutamine from the BoNT/A cleavage site scissile bond from a SNAP-25 cleavage product or .alpha.-SNAP-25 polyclonal antibodies that can bind an epitope comprising a carboxyl-terminus glutamine from the BoNT/A cleavage site scissile bond from a SNAP-25 cleavage product.

[0019] Still another aspect disclosed in the present specification provides .alpha.-SNAP-25 antibodies that can bind an epitope comprising a carboxyl-terminus at the P.sub.1 residue from the BoNT/A cleavage site scissile bond from a SNAP-25 cleavage product. Such .alpha.-SNAP-25 antibodies include both naturally-occurring and non-naturally-occurring antibodies, as well as, monoclonal .alpha.-SNAP-25 antibodies or polyclonal .alpha.-SNAP-25 antibodies. Monoclonal .alpha.-SNAP-25 antibodies useful as .alpha.-SNAP-25 antibodies that bind an epitope comprising a carboxyl-terminus at the P.sub.1 residue from the BoNT/A cleavage site scissile bond from a SNAP-25 cleavage product, include, without limitation, the monoclonal .alpha.-SNAP-25 antibodies produced from hybridoma cell lines 1D3B8, 2C9B10, 2E2A6, 3C1A5 and 3C3E2.

[0020] Yet another aspect disclosed in the present specification provides methods of detecting BoNT/A activity. Aspects of this method comprise the steps of (a) treating a cell from an established cell line with a sample comprising a BoNT/A, wherein the cell from an established cell line is susceptible to BoNT/A intoxication; (b) isolating from the treated cell a SNAP-25 component comprising a SNAP-25 cleavage product having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond; (c) contacting the SNAP-25 component with an .alpha.-SNAP-25 antibody that can bind an epitope comprising a carboxyl-terminus at the P.sub.1 residue from the BoNT/A cleavage site scissile bond from a SNAP-25 cleavage product; and (d) detecting the presence of an antibody-antigen complex comprising the .alpha.-SNAP-25 antibody and the SNAP-25 cleavage product; wherein detection by the antibody-antigen complex is indicative of BoNT/A activity. The .alpha.-SNAP-25 antibody of step c can optionally be linked to a solid phase support.

[0021] Yet another aspect disclosed in the present specification provides methods of detecting BoNT/A activity. Aspects of this method comprise the steps of (a) treating a cell from an established cell line with a sample comprising a BoNT/A, wherein the cell from an established cell line can uptake a BoNT/A; (b) isolating from the treated cell a SNAP-25 component comprising a SNAP-25 having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond; (c) contacting the SNAP-25 component with an .alpha.-SNAP-25 antibody that can bind an epitope comprising a carboxyl-terminus at the P.sub.1 residue from the BoNT/A cleavage site scissile bond from a SNAP-25 cleavage product; and (d) detecting the presence of an antibody-antigen complex comprising the .alpha.-SNAP-25 antibody and the SNAP-25 cleavage product; wherein detection by the antibody-antigen complex is indicative of BoNT/A activity. The .alpha.-SNAP-25 antibody of step c can optionally be linked to a solid phase support.

[0022] A further aspect disclosed in the present specification provides methods of determining BoNT/A immunoresistance in a mammal. Aspects of this method comprise the steps of (a) adding a BoNT/A to a test sample obtained from a mammal being tested for the presence or absence of .alpha.-BoNT/A neutralizing antibodies; (b) treating a cell from an established cell line with the test sample, wherein the cell from an established cell line is susceptible to BoNT/A intoxication; (c) isolating from the treated cells a SNAP-25 component comprising a SNAP-25 cleavage product having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond; (d) contacting the SNAP-25 component with an .alpha.-SNAP-25 antibody that can bind an epitope comprising a carboxyl-terminus at the P.sub.1 residue from the BoNT/A cleavage site scissile bond from a SNAP-25 cleavage product; (e) detecting the presence of an antibody-antigen complex comprising the .alpha.-SNAP-25 antibody and the SNAP-25 cleavage product; (f) repeating steps a-e with a negative control sample instead of a test sample; and (g) comparing the amount of antibody-antigen complex detected in step (e) to the amount of antibody-antigen complex detected in step (f), wherein detection of a lower amount of antibody-antigen complex detected in step (e) relative to the amount of antibody-antigen complex detected in step (f) is indicative of the presence of .alpha.-BoNT/A neutralizing antibodies. The .alpha.-SNAP-25 antibody of step d can optionally be linked to a solid phase support. The control sample in step f can also include a positive control sample, in addition to the negative control sample.

[0023] Clostridia toxins produced by Clostridium botulinum, Clostridium tetani, Clostridium baratii and Clostridium butyricum are the most widely used in therapeutic and cosmetic treatments of humans and other mammals. Strains of C. botulinum produce seven antigenically-distinct serotypes of botulinum toxins (BoNTs), which have been identified by investigating botulism outbreaks in man (BoNT/A, BoNT/B, BoNT/E and BoNT/F), animals (BoNT/C1 and BoNT/D), or isolated from soil (BoNT/G). While all seven botulinum toxin serotypes have similar structure and biological properties, each also displays heterogeneous characteristics, such as, e.g., different pharmacological properties. In contrast, tetanus toxin (TeNT) is produced by a uniform group of C. tetani. Two other species of Clostridia, C. baratii and C. butyricum, also produce toxins similar to BoNT/F and BoNT/E, respectively.

[0024] Clostridial toxins are each translated as a single chain polypeptide of approximately 150 kDa that is subsequently cleaved by proteolytic scission within a disulfide loop by a naturally-occurring protease, such as, e.g., an endogenous Clostridial toxin protease or a naturally-occurring protease produced in the environment. This posttranslational processing yields a di-chain molecule comprising an approximately 50 kDa light chain (LC) and an approximately 100 kDa heavy chain (HC) held together by a single disulfide bond and noncovalent interactions. Each mature di-chain molecule comprises three functionally distinct domains: 1) an enzymatic domain located in the LC that includes a metalloprotease region containing a zinc-dependent endopeptidase activity which specifically targets core components of the neurotransmitter release apparatus; 2) a translocation domain contained within the amino-terminal half of the HC (H.sub.N) that facilitates release of the LC from intracellular vesicles into the cytoplasm of the target cell; and 3) a binding domain found within the carboxyl-terminal half of the HC (H.sub.C) that determines the binding activity and binding specificity of the toxin to the receptor complex located at the surface of the target cell.

[0025] The binding, translocation and enzymatic activity of these three functional domains are all necessary for toxicity. While all details of this process are not yet precisely known, the overall cellular intoxication mechanism whereby Clostridial toxins enter a neuron and inhibit neurotransmitter release is similar, regardless of serotype or subtype. Although the applicants have no wish to be limited by the following description, the intoxication mechanism can be described as comprising at least four steps: 1) receptor binding, 2) complex internalization, 3) light chain translocation, and 4) enzymatic target modification (FIGS. 1A-1B). The process is initiated when the HC domain of a Clostridial toxin binds to a toxin-specific receptor system located on the plasma membrane surface of a target cell. The binding specificity of a receptor complex is thought to be achieved, in part, by specific combinations of gangliosides and protein receptors that appear to distinctly comprise each Clostridial toxin receptor complex. Once bound, the toxin/receptor complexes are internalized by endocytosis and the internalized vesicles are sorted to specific intracellular routes. The translocation step appears to be triggered by the acidification of the vesicle compartment. This process seems to initiate important pH-dependent structural rearrangements that increase hydrophobicity, promote pore formation, and facilitate separation of the heavy and light chains of the toxin. Once separated, the light chain endopeptidase of the toxin is released from the intracellular vesicle into the cytosol where it appears to specifically target core components of the neurotransmitter release apparatus. These core proteins, vesicle-associated membrane protein (VAMP)/synaptobrevin, synaptosomal-associated protein of 25 kDa (SNAP-25) and Syntaxin, are necessary for synaptic vesicle docking and fusion at the nerve terminal and constitute members of the soluble N-ethylmaleimide-sensitive factor-attachment protein-receptor (SNARE) family. BoNT/A and BoNT/E cleave SNAP-25 in the carboxyl terminal region, releasing a nine or twenty six amino acid fragment, respectively, and BoNT/C1 also cleaves SNAP-25 near the carboxyl terminus releasing an eight amino acid fragment. The botulinum serotypes BoNT/B, BoNT/D, BoNT/F and BoNT/G, and tetanus toxin, act on the conserved central portion of VAMP, and release the amino terminal portion of VAMP into the cytosol. BoNT/C1 cleaves syntaxin at a single site near the cytosolic membrane surface. The selective proteolysis of synaptic SNAREs accounts for the block of neurotransmitter release caused by Clostridial toxins in vivo. The SNARE protein targets of Clostridial toxins are common to exocytosis in a variety of non-neuronal types; in these cells, as in neurons, light chain peptidase activity inhibits exocytosis, see, e.g., Yann Humeau et al., How Botulinum and Tetanus Neurotoxins Block Neurotransmitter Release, 82(5) Biochimie. 427-446 (2000); Kathryn Turton et al., Botulinum and Tetanus Neurotoxins: Structure, Function and Therapeutic Utility, 27(11) Trends Biochem. Sci. 552-558. (2002); Giovanna Lalli et al., The Journey of Tetanus and Botulinum Neurotoxins in Neurons, 11(9) Trends Microbiol. 431-437, (2003).

[0026] Aspects of the present disclosure comprise, in part, a composition for producing .alpha.-SNAP-25 antibodies that can bind an epitope comprising a carboxyl-terminus at the P.sub.1 residue from the BoNT/A cleavage site scissile bond from a SNAP-25 cleavage product. Other aspects of the present disclosure comprise, in part, an immune response inducing composition for producing .alpha.-SNAP-25 antibodies that can bind an epitope comprising a carboxyl-terminus at the P.sub.1 residue from the BoNT/A cleavage site scissile bond from a SNAP-25 cleavage product. As used herein, the term "immune response inducing composition" refers to a composition comprising a SNAP-25 antigen which, when administered to an animal, stimulates an immune response against the SNAP-25 antigen, thereby producing .alpha.-SNAP-25 antibodies that can bind an epitope comprising a carboxyl-terminus at the P.sub.1 residue from the BoNT/A cleavage site scissile bond from a SNAP-25 cleavage product. The term "immune response" refers to any response by the immune system of an animal to an immune response inducing composition. Exemplary immune responses include, but are not limited to, cellular as well as local and systemic humoral immunity, such as, e.g., CTL responses, including antigen-specific induction of CD8+ CTLs, helper T-cell responses, including T-cell proliferative responses and cytokine release, and B-cell responses including, e.g., an antibody producing response. The term "inducing an immune response" refers to administration of an immune response inducing composition or a polynucleotide encoding the immune response inducing composition, where an immune response is affected, i.e., stimulated, initiated or induced.

[0027] A composition comprises a SNAP-25 antigen. As used herein, the term "antigen" refers to a molecule that elicits an immune response and includes, without limitation, peptides, polysaccharides and conjugates of lipids, such as, e.g., lipoproteins and glycolipids. As used herein, the term "SNAP-25 antigen" refers to any antigen which has a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond that can elicit an immune response. A SNAP-25 antigen used in an immune response inducing composition must be large enough to be substantially unique in sequence, thus reducing the possibility of producing antibodies that are cross reactive against antigens other than SNAP-25. In addition, a SNAP-25 antigen used in an immune response inducing composition must be small enough to only trigger an immune response substantially against a SNAP-25 having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond, thus increasing the possibility of producing .alpha.-SNAP-25 antibodies that can distinguish a SNAP-25 having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond from a SNAP-25 lacking a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond. Furthermore, it is also very desirable to generate .alpha.-SNAP-25 antibodies of a single amino acid sequence in a good yield that are reproducibly selective and which bind with acceptable avidity in order to permit the design of a highly sensitive assay.

[0028] The sequence surrounding a BoNT/A cleavage site present in SNAP-25 is denoted as -P.sub.5-P.sub.4-P.sub.3-P.sub.2-P.sub.1-P.sub.1'-P.sub.2'-P.sub.3'-P.sub- .4'-P.sub.5', with P.sub.1-P.sub.1' representing the scissile bond. Upon cleavage by BoNT/A, the resulting cleavage products produced comprise a fragment including the P.sub.5-P.sub.4-P3-P2-P1 sequence and a fragment including the P1'-P2'-P3'-P4'-P5'. Thus, as used herein, the term "SNAP-25 having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond" refers to any SNAP-25 having the P.sub.1 residue as its carboxyl-terminal amino acid. For example, Q.sub.197-R.sub.198 of human SNAP-25 (SEQ ID NO: 5) represents the P.sub.1-P.sub.1' scissile bond for the BoNT/A cleavage site. As such, "SNAP-25 having a carboxyl-terminus glutamine of the BoNT/A cleavage site scissile bond" would be any SNAP-25 cleavage product having a glutamine at its carboxyl-terminal amino acid where the glutamine represents Q.sub.197 of the scissile bond. As another example, K.sub.204-H.sub.205 of Torpedo marmorata SNAP-25 (SEQ ID NO: 16) represents the P.sub.1-P.sub.1' scissile bond for the BoNT/A cleavage site. As such, "SNAP-25 having a carboxyl-terminus lysine of the BoNT/A cleavage site scissile bond" would be any SNAP-25 cleavage product having a lysine at its carboxyl-terminal amino acid where the lysine represents K.sub.204 of the scissile bond.

[0029] The SNAP-25 antigen having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond from the BoNT/A cleavage site can be modified to enhance the immunogenicity of a SNAP-25 antigen, a hapten, or any other antigenic compound that is immunogenic, non-immunogenic, or weakly immunogenic when not associated with the modification. In an aspect of this embodiment, the carboxyl-terminal P.sub.1 residue from the scissile bond of a SNAP-25 antigen can be carboxylated. Carboxylation increases the desired immunogenic properties of a SNAP-25 antigen in two respects. First, because charged amino acids enhance immunogenicity, adding a COO.sup.- group to the carboxyl-terminal residue will increase the overall immunogenicity of a SNAP-25 antigen. Second, because the P.sub.1 residue of the BoNT/A cleavage site scissile bond is in a charged state upon cleavage, adding a COO.sup.- group to the carboxyl-terminal residue will better mimic the actual antigen that the .alpha.-SNAP-25 antibodies disclosed in the present specification are designed to bind.

[0030] In an aspect of this embodiment, the amino-terminal residue from a SNAP-25 antigen can be modified by the addition of an amino acid adapted to attach the SNAP-25 antigen to a carrier protein, such as, e.g., a keyhole limpet hemacyanin (KLH), an ovalbumin (OVA), a thyroglobulin (THY), a bovine serum albumin (BSA), a soybean trypsin inhibitor (STI), or a multiple attachment peptide (MAP). For example, a cysteine residue can be placed at the amino-terminus in order to conjugate the carrier protein KLH.

[0031] Thus, an embodiment, a SNAP-25 antigen having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond can be, e.g., at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, or at least 30 amino acids in length. In another embodiment, a SNAP-25 antigen having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond can be, e.g., at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 25, or at most 30 amino acids in length. In still another embodiment, a SNAP-25 antigen having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond can be, e.g., between 7-12 amino acids, between 10-15 amino acids, or between 13-18 amino acids.

[0032] In another embodiment, the SNAP-25 antigen having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond comprises SEQ ID NO: 32. In aspects of this embodiment, the SNAP-25 antigen having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond comprises SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 147 or SEQ ID NO: 148. In a further embodiment, the SNAP-25 antigen having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond comprises SEQ ID NO: 38.

[0033] In yet another embodiment, the SNAP-25 antigen having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond comprises SEQ ID NO: 39. In aspects of this embodiment, the SNAP-25 antigen having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond comprises SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43 or SEQ ID NO: 44. In a further embodiment, the SNAP-25 antigen having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond comprises SEQ ID NO: 45.

[0034] It is envisioned that any and all SNAP-25 antigens that triggers an immune response that produces .alpha.-SNAP-25 antibodies that bind an epitope comprising a carboxyl-terminus at the P.sub.1 residue from the BoNT/A cleavage site scissile bond from a SNAP-25 cleavage product can be useful as a SNAP-25 antigen. Thus, amino acid sequence variants comprising SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 147 or SEQ ID NO: 148 can be useful as a SNAP-25 antigen to trigger an immune response that produces .alpha.-SNAP-25 antibodies that bind an epitope comprising a carboxyl-terminus at the P.sub.1 residue from the BoNT/A cleavage site scissile bond from a SNAP-25 cleavage product. Thus, in an embodiment, a SNAP-25 antigen can substitute at least 1, at least 2, at least 3, at least 4, or at least 5 amino acid substitutions, deletions or additions to the SNAP-25 antigens comprising SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 147 or SEQ ID NO: 148. In still another embodiment, a SNAP-25 antigen can have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% amino acid identity to the SNAP-25 antigens comprising SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 147 or SEQ ID NO: 148.

[0035] It is envisioned that one or more carriers may be linked to a SNAP-25 antigen in order to enhance the immunogenicity of a SNAP-25 antigen that is immunogenic, non-immunogenic, or weakly immunogenic when not associated with the carrier. Non-limiting examples, include, e.g., a keyhole limpet hemacyanin (KLH), an ovalbumin (OVA), a thyroglobulin (THY), a bovine serum albumin (BSA), a soybean trypsin inhibitor (STI), or a multiple attachment peptide (MAP). As is well known in the art, a non-antigenic or weakly antigenic antigen can be made antigenic by coupling the antigen to a carrier. Various other carrier and methods for coupling an antigen to a carrier are well known in the art. See, e.g., Harlow and Lane, supra, 1998a; Harlow and Lane, supra, 1998b; and David W. Waggoner, Jr. et al., Immunogenicity-enhancing carriers and compositions thereof and methods of using the same, U.S. Patent Publication No. 20040057958 (Mar. 25, 2004). An epitope can also be generated by expressing the epitope as a fusion protein. Methods for expressing polypeptide fusions are well known to those skilled in the art as described, for example, in Ausubel et al., Current Protocols in Molecular Biology (Supplement 47), John Wley & Sons, New York (1999). As the carboxyl-terminal end of the SNAP-25 antigen must be the P.sub.1 residue of the BoNT/A cleavage site scissile bond, a carrier must be linked to the amino end of the SNAP-25 antigen.

[0036] It is envisioned that one or more flexible spacers may be linked to a SNAP-25 antigen in order to enhance the immunogenicity of a SNAP-25 antigen that is immunogenic, non-immunogenic, or weakly immunogenic when not associated with the flexible linkers. A flexible spacer increases the overall peptide length of the SNAP-25 antigen and provides flexibility, thereby facilitating the proper presentation of the SNAP-25 antigen to the immune cells. As a non-limiting example, a composition can comprise a SNAP-25 antigen linked to one or more flexible spacers in tandem to better present SNAP-25 antigen to immune cells, thereby facilitating the immune response.

[0037] A flexible space comprising a peptide is at least one amino acid in length and comprises non-charged amino acids with small side-chain R groups, such as, e.g., glycine, alanine, valine, leucine or serine. Thus, in an embodiment a flexible spacer can be, e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 amino acids in length. In another embodiment, a flexible spacer can be, e.g., at least 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, or at most 10 amino acids in length. In still another embodiment, a flexible spacer can be, e.g., between 1-3 amino acids, between 2-4 amino acids, between 3-5 amino acids, between 4-6 amino acids, or between 5-7 amino acids. Non-limiting examples of a flexible spacer include, e.g., a G-spacers such as GGG, GGGG (SEQ ID NO: 55), and GGGGS (SEQ ID NO: 56) or an A-spacers such as AAA, AAAA (SEQ ID NO: 57) and AAAAV (SEQ ID NO: 58). A flexible spacer is linked in-frame to the SNAP-25 antigen as a fusion protein.

[0038] As discussed above, a flexible spacer is used, in part, to increase the overall peptide length of the SNAP-25 antigen. For example, a 5-10 amino acid SNAP-25 antigen can have its overall length increased by linking a 3-5 amino acid flexible space to the amino-end of the SNAP-25 antigen. As another example, a 5-10 amino acid SNAP-25 antigen can have its overall length increased by linking a 4-6 amino acid flexible space to the amino-end of the SNAP-25 antigen. As another example, a 5-10 amino acid SNAP-25 antigen can have its overall length increased by linking a 7-10 amino acid flexible space to the amino-end of the SNAP-25 antigen. As another example, a 7-12 amino acid SNAP-25 antigen can have its overall length increased by linking a 1-3 amino acid flexible space to the amino-end of the SNAP-25 antigen. As another example, a 7-12 amino acid SNAP-25 antigen can have its overall length increased by linking a 4-6 amino acid flexible space to the amino-end of the SNAP-25 antigen. The increased length provided by the flexible spacer allows for the selection of a small sized SNAP-25 antigen, thereby increasing the likelihood that the SNAP-25 antigen will only trigger an immune response substantially against a SNAP-25 having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond, thus increasing the possibility of producing .alpha.-SNAP-25 antibodies that can distinguish a SNAP-25 having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond from a SNAP-25 lacking a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond.

[0039] It is envisioned that compositions disclosed in the present specification can optionally comprise a SNAP-25 antigen disclosed in the present specification and one or more adjuvants. As used herein, the term "adjuvant" when used in reference to a SNAP-25 composition refers to any substance or mixture of substances that increases or diversifies the immune response to a SNAP-25 antigen. An adjuvant can, for example, serve to reduce the number of immunizations or the amount of antigen required for protective immunization. The use of adjuvants in an immune response inducing composition is well known. The main objective of these adjuvants is to allow an increase in the immune response. Non-limiting adjuvants include, e.g., liposomes, oily phases, including, without limitation, the Freund type of adjuvants, such as, e.g., Freund's complete adjuvant (FCA); Freund's incomplete adjuvant (FIA); sapogenin glycosides, such as, e.g., saponins; carbopol; N-acetylmuramyl-L-alanyl-D-isoglutamine (commonly known as muramyl dipeptide or "MDP"); and lipopolysaccharide (LPS). Such adjuvants are generally used in the form of an emulsion with an aqueous phase, or, more commonly, may consist of water-insoluble inorganic salts. These inorganic salts may consist, for example, of aluminum hydroxide, zinc sulfate, colloidal iron hydroxide, calcium phosphate or calcium chloride. Aluminum hydroxide (Al(OH).sub.3) is a commonly used adjuvant. Currently, the only FDA-approved adjuvant for use in humans is aluminum salts (Alum) which are used to "depot" antigens by precipitation of the antigens. Adjuvants provided above are merely exemplary. In fact, any adjuvant may be used in a SNAP-25 composition disclosed in the present specification as long as the adjuvant satisfies the requisite characteristics for inducing an immune response.

[0040] A carrier disclosed in the present specification may also act as an adjuvant. Specific adjuvants and methods of making and using are described in, e.g., Gupta et al. Vaccine, 11: 993-306, 1993; Arnon, R. (Ed.) Synthetic Vaccines 1:83-92, CRC Press, Inc., Boca Raton, Fla., 1987; and David W. Waggoner, Jr. et al., Immunogenicity-Enhancing Carriers and Compositions Thereof and Methods of Using the Same, U.S. Patent Publication No. 20040057958 (Mar. 25, 2004). Additional adjuvants include any compound described in Chapter 7 (pp 141-227) of "Vaccine Design, The Subunit and Adjuvant Approach" (eds. Powell, M. F. and Newman, M. J.) Pharmaceutical Biotechnology, Volume 6, Plenum Press (New York). Examples from this compendium include Muramyl Dipeptide (MDP) and Montanide 720. Molecules such as Poly lnosine:Cytosine (Poly I:C) or plasmid DNA containing CpG motifs can also be administered as adjuvants in combination with antigens encapsulated in microparticles. In another example, the adjuvant is an agent that facilitates entry of the antigenic compound into the cytoplasm of a cell such as listeriolysin, streptolysin or a mixture thereof.

[0041] Thus, in an embodiment, a SNAP-25 composition comprises a SNAP-25 antigen having a carboxylated carboxyl-terminal glutamine linked to a carrier peptide. In aspects of this embodiment, a SNAP-25 antigen having a carboxylated carboxyl-terminal glutamine comprises SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 147 or SEQ ID NO: 148. In another aspect of this embodiment, a SNAP-25 antigen comprises SEQ ID NO: 38. In aspects of this embodiment, the carrier peptide is a keyhole limpet hemacyanin (KLH), an ovalbumin (OVA), a thyroglobulin (THY), a bovine serum albumin (BSA), a soybean trypsin inhibitor (STI) or a multiple attachment peptide (MAP).

[0042] In another embodiment, a SNAP-25 composition comprises a SNAP-25 antigen having a carboxylated carboxyl-terminal lysine linked to a carrier peptide. In aspects of this embodiment, SNAP-25 antigen having a carboxylated carboxyl-terminal lysine comprises SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43 or SEQ ID NO: 44. In another aspect of this embodiment, a SNAP-25 antigen comprises SEQ ID NO: 45. In aspects of this embodiment, the carrier peptide is a keyhole limpet hemacyanin (KLH), an ovalbumin (OVA), a thyroglobulin (THY), a bovine serum albumin (BSA), a soybean trypsin inhibitor (STI) or a multiple attachment peptide (MAP).

[0043] In yet another embodiment, a SNAP-25 composition comprises a SNAP-25 antigen having a carboxylated C-terminal glutamine linked to one or more flexible linkers and a carrier peptide wherein the flexible linkers intervene between the SNAP-25 antigen and the carrier peptide. In aspects of this embodiment, SNAP-25 antigen having a carboxylated carboxyl-terminal glutamine comprises SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 147 or SEQ ID NO: 148. In another embodiment, a SNAP-25 antigen comprises SEQ ID NO: 46. In aspects of this embodiment, the carrier peptide is a keyhole limpet hemacyanin (KLH), an ovalbumin (OVA), a thyroglobulin (THY), a bovine serum albumin (BSA), a soybean trypsin inhibitor (STI) or a multiple attachment peptide (MAP). In aspects of this embodiment, the flexible linker is a G-spacer or an A-spacer.

[0044] In still another embodiment, a SNAP-25 composition comprises a SNAP-25 antigen having a carboxylated C-terminal lysine linked to a flexible linker and a carrier peptide wherein the flexible linker intervenes between the SNAP-25 antigen and the carrier peptide. In aspects of this embodiment, SNAP-25 antigen having a carboxylated carboxyl-terminal lysine comprises SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43 or SEQ ID NO: 44. In another aspect of this embodiment, a SNAP-25 antigen comprises SEQ ID NO: 47. In aspects of this embodiment, the carrier peptide is a keyhole limpet hemacyanin (KLH), an ovalbumin (OVA), a thyroglobulin (THY), a bovine serum albumin (BSA), a soybean trypsin inhibitor (STI) or a multiple attachment peptide (MAP). In aspects of this embodiment, the flexible linker is a G-spacer or an A-spacer.

[0045] Aspects of the present disclosure comprise, in part, a method for producing .alpha.-SNAP-25 antibodies that bind an epitope comprising a carboxyl-terminus at the P.sub.1 residue from the BoNT/A cleavage site scissile bond from a SNAP-25 cleavage product. An .alpha.-SNAP-25 antibody that binds an epitope comprising a carboxyl-terminus at the P.sub.1 residue from the BoNT/A cleavage site scissile bond from a SNAP-25 cleavage product can be produced by a wide variety of methods that are well known in the art. Specific protocols for making and using antibodies as well as detecting, and measuring antibody binding specificity, binding affinity and binding avidity are known in the art. See, e.g., ANTIBODIES: A LABORATORY MANUAL (Edward Harlow & David Lane, eds., Cold Spring Harbor Laboratory Press, 2.sup.nd ed. 1998a); and USING ANTIBODIES: A LABORATORY MANUAL: PORTABLE PROTOCOL No. I (Edward Harlow & David Lane, Cold Spring Harbor Laboratory Press, 1998b); Molecular Cloning, A Laboratory Manual, 2001; and Current Protocols in Molecular Biology, 2004; David Anderson et al., Therapeutic Polypeptides, Nucleic Acids Encoding Same, and Methods of Use, U.S. Pat. No. 7,034,132 (Apr. 25, 2005); and Beatriz M. Carreno et al., Antibodies Against CTLA4, U.S. Pat. No. 7,034,121 (Apr. 25, 2006).

[0046] As a non-limiting example, .alpha.-SNAP-25 polyclonal antibodies that bind an epitope comprising a carboxyl-terminus at the P.sub.1 residue from the BoNT/A cleavage site scissile bond from a SNAP-25 cleavage product can be produced by injecting an animal, such as, e.g., a rabbit, a goat, a mouse or another mammal, with one or more injections of a composition disclosed in the present specification. As another non-limiting example, .alpha.-SNAP-25 polyclonal antibodies that bind an epitope comprising a carboxyl-terminus at the P.sub.1 residue from the BoNT/A cleavage site scissile bond from a SNAP-25 cleavage product can be produced by injecting an egg, such as, e.g., a chicken egg, with one or more injections of a composition disclosed in the present specification. The antibody titer in the immunized animal can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized antigen or a cell-based activity assay. If desired, polyclonal antibodies for an .alpha.-SNAP-25 antibody that binds an epitope comprising a carboxyl-terminus at the P.sub.1 residue from the BoNT/A cleavage site scissile bond from a SNAP-25 cleavage product can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as protein A affinity chromatography to obtain the IgG fraction, or by affinity purification against the peptide used for producing the antibodies.

[0047] As another non-limiting example, .alpha.-SNAP-25 monoclonal antibody that binds an epitope comprising a carboxyl-terminus at the P.sub.1 residue from the BoNT/A cleavage site scissile bond from a SNAP-25 cleavage product can be produced using a hybridoma method. See e.g., Chapter 6 Monoclonal Antibodies, pp. 196-244, Harlow & Lane, supra, 1998a; and Chapter 7 Growing Hybridomas, pp. 245-282, Harlow & Lane, supra, 1998a; and Goding, pp. 59-103, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986). In this method, a host animal, such as, e.g., a mouse, a hamster, or another appropriate host animal, is typically exposed to one or more injections of a SNAP-25 antigen disclosed in the present specification to elicit lymphocytes that produce or are capable of producing .alpha.-SNAP-25 antibodies that will specifically bind to a SNAP-25 having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond. The antibody titer in the immunized animal can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized antigen or a cell-based activity assay. Alternatively, the lymphocytes can be immunized in vitro using a suitable cell culture line. At an appropriate time after immunization, e.g., when the antibody titers are highest, antibody-producing cells are isolated from the animal. Generally, either peripheral blood lymphocytes are used, if cells of human origin are desired, or spleen cells or lymph node cells are used, if non-human mammalian sources are desired. The isolated antibody-producing cells are fused with an immortal cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell. Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Typically, a murine myeloma cell line is fused with splenocytes harvested from an appropriately immunized mouse to produce the hybridoma. Preferred immortal cell lines are mouse myeloma cell lines that are sensitive to culture medium containing hypoxanthine, aminopterin and thymidine (HAT). Any of a number of myeloma cell lines can be used as a fusion partner according to standard techniques, e.g., the P3-NS1/1-Ag4-1, P3-x63-Ag8.653 or Sp2/O--Ag14 myeloma lines. Hybridoma cells resulting from the fusion are then selected using HAT medium, which kills unfused and unproductively fused myeloma cells (unfused splenocytes die after several days in culture because they are not transformed). The culture medium in which the hybridoma cells are grown can then be assayed for the presence of .alpha.-SNAP-25 monoclonal antibodies that bind an epitope comprising a carboxyl-terminus at the P.sub.1 residue from the BoNT/A cleavage site scissile bond from a SNAP-25 cleavage product. For example, hybridoma supernatants can be screened using .alpha.-SNAP-25 positive media in an immunoprecipitation assay, in vitro binding assay, such as, e.g., a radioimmunoassay (RIA) or an enzyme-linked immunoabsorbent assay (ELISA), or in a cell-based activity assay. Such techniques and assays are known in the art. See e.g., Chapter 11 Immunoprecipitation, pp. 421-470, Harlow & Lane, supra, 1998a; Chapter 12 Immunoblotting, pp. 471-510, Harlow & Lane, supra, 1998a; Chapter 14 Immunoassays, pp. 553-612, Harlow & Lane, supra, 1998a. Additional studies can then be done to determine whether the antibody is also unreactive to a SNAP-25 lacking a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond. The binding affinity of an .alpha.-SNAP-25 monoclonal antibody can also be determined, e.g., by Scatchard analysis. See, e.g., Peter J. Munson and David Rodbard, Ligand: A Versatile Computerized Approach For Characterization of Ligand-Binding Systems, 107(1) Anal. Biochem. 220-239 (1980). After the desired hybridoma cells are identified, limiting dilution procedures are used to isolate clones originating from a single cell until a clonal cell line expressing the desired monoclonal antibody is obtained. Those antibodies sufficiently selective for a SNAP-25 having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond and bind with sufficiently high avidity are chosen for further characterization and study.

[0048] Another alternative for preparing an .alpha.-SNAP-25 monoclonal antibody that binds an epitope comprising a carboxyl-terminus at the P.sub.1 residue from the BoNT/A cleavage site scissile bond from a SNAP-25 cleavage product is by screening a recombinant combinatorial immunoglobulin library, such as, e.g., an antibody phage display library, with a SNAP-25 peptide and isolate immunoglobulin library members that bind a SNAP-25 having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond. Kits for generating and screening phage display libraries are commercially available, such as, e.g., the Recombinant Phage Antibody System (Amersham GE Healthcare, Piscataway, N.J.); and the SurfZAP.TM. Phage Display Kit (Stratagene, La Jolla, Calif.). Additionally, examples of methods and reagents useful in generating and screening antibody display library can be found in, for example, Ladner et al. U.S. Pat. No. 5,223,409; Borrebaeck et al. U.S. Pat. No. 5,712,089; Griffiths et al. U.S. Pat. No. 5,885,793; Griffiths et al. U.S. Pat. No. 5,962,255; McCafferty et al. U.S. Pat. No. 5,969,108; Griffiths et al. U.S. Pat. No. 6,010,884; Jespers et al. U.S. Pat. No. 6,017,732; Borrebaeck et al. U.S. Pat. No. 6,027,930; Johnson et al. U.S. Pat. No. 6,140,471; McCafferty et al. U.S. Pat. No. 6,172,197, each of which is hereby incorporated by reference in its entirety.

[0049] Aspects of the present disclosure comprise, in part, collecting a sample containing an .alpha.-SNAP-25 antibody or .alpha.-SNAP-25 antibody-producing cells. As used herein, the term "sample containing an .alpha.-SNAP-25 antibody or .alpha.-SNAP-25 antibody-producing cell" refers to any biological matter that contains or potentially contains at least one an .alpha.-SNAP-25 antibody that that bind an epitope comprising a carboxyl-terminus at the P.sub.1 residue from the BoNT/A cleavage site scissile bond from a SNAP-25 cleavage product. It is envisioned that any and all samples that can contain an .alpha.-SNAP-25 antibody that binds an epitope comprising a carboxyl-terminus at the P.sub.1 residue from the BoNT/A cleavage site scissile bond from a SNAP-25 cleavage product can be used in this method, including, without limitation, blood, plasma, serum and lymph fluid. It is also envisioned that any cell capable of producing an .alpha.-SNAP-25 antibody that binds an epitope comprising a carboxyl-terminus at the P.sub.1 residue from the BoNT/A cleavage site scissile bond from a SNAP-25 cleavage product can be used in this method, including, without limitation, a CD8 cells, a CTL cell, a helper T-cell and a B-cell. A variety of well known methods can be used for collecting from an individual a sample containing the .alpha.-SNAP-25 antibody or .alpha.-SNAP-25 antibody-producing cell, see, e.g., Harlow & Lane, supra, 1998a; and Harlow & Lane, supra, 1998b. Similarly, a variety of well known methods can be used for processing a sample to isolate an .alpha.-SNAP-25 antibody that binds an epitope comprising a carboxyl-terminus at the P.sub.1 residue from the BoNT/A cleavage site scissile bond from a SNAP-25 cleavage product. A procedure for collecting a sample can be selected based on the type of antibody to be isolated. As a non-limiting example, when isolating an .alpha.-SNAP-25 polyclonal antibodies that bind an epitope comprising a carboxyl-terminus at the P.sub.1 residue from the BoNT/A cleavage site scissile bond from a SNAP-25 cleavage product, an appropriate sample can be a blood sample containing such .alpha.-SNAP-25 antibodies, whereas when isolating an .alpha.-SNAP-25 monoclonal antibodies that bind an epitope comprising a carboxyl-terminus at the P.sub.1 residue from the BoNT/A cleavage site scissile bond from a SNAP-25 cleavage product, an appropriate sample can be an .alpha.-SNAP-25 antibody-producing cell such as a spleen cell or hybridoma.

[0050] Aspects of the present disclosure comprise, in part, isolating an .alpha.-SNAP-25 antibody that binds an epitope comprising a carboxyl-terminus at the P.sub.1 residue from the BoNT/A cleavage site scissile bond from a SNAP-25 cleavage product from the sample. Methods of isolating an such .alpha.-SNAP-25 antibodies, such as, e.g., .alpha.-SNAP-25 polyclonal antibodies that bind an epitope comprising a carboxyl-terminus at the P.sub.1 residue from the BoNT/A cleavage site scissile bond from a SNAP-25 cleavage product or .alpha.-SNAP-25 monoclonal antibodies that bind an epitope comprising a carboxyl-terminus at the P1 residue from the BoNT/A cleavage site scissile bond from a SNAP-25 cleavage product are well known to those skilled in the art. See, e.g., Harlow and Lane, supra, 1998a; and Harlow and Lane, supra, 1998b. For example, such .alpha.-SNAP-25 polyclonal antibodies can be isolated from the sample by well known techniques, such as, e.g., affinity chromatography using protein A or protein G, which provide primarily the IgG fraction of immune serum. Subsequently, or alternatively, a specific SNAP-25 antigen can be immobilized on a column or magnetic beads to purify the .alpha.-SNAP-25 polyclonal antibodies that bind an epitope comprising a carboxyl-terminus at the P.sub.1 residue from the BoNT/A cleavage site scissile bond from a SNAP-25 cleavage product by immunoaffinity chromatography. An .alpha.-SNAP-25 monoclonal antibody that binds an epitope comprising a carboxyl-terminus at the P.sub.1 residue from the BoNT/A cleavage site scissile bond from a SNAP-25 cleavage product can be isolated from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, e.g., protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0051] Thus, in an embodiment, a method of producing an .alpha.-SNAP-25 antibody that binds an epitope comprising a carboxyl-terminus at the P.sub.1 residue from the BoNT/A cleavage site scissile bond from a SNAP-25 cleavage product comprises the steps (a) administering to an animal a composition comprising a SNAP-25 antigen having a carboxylated C-terminal glutamine linked to a carrier peptide; (b) collecting from the animal a sample containing an .alpha.-SNAP-25 antibody or .alpha.-SNAP-25 antibody-producing cell; and (c) isolating the .alpha.-SNAP-25 antibody component from the sample. In an aspect of this embodiment, the .alpha.-SNAP-25 antibody that binds an epitope comprising a carboxyl-terminus at the P.sub.1 residue from the BoNT/A cleavage site scissile bond from a SNAP-25 cleavage product is a polyclonal antibody. In another aspect of this embodiment, an .alpha.-SNAP-25 antibody that binds an epitope comprising a carboxyl-terminus at the P.sub.1 residue from the BoNT/A cleavage site scissile bond from a SNAP-25 cleavage product is a monoclonal antibody. In a further aspect of this embodiment, an .alpha.-SNAP-25 monoclonal antibody that binds an epitope comprising a carboxyl-terminus at the P.sub.1 residue from the BoNT/A cleavage site scissile bond from a SNAP-25 cleavage product produced is an IgG subtype. In other aspects of this embodiment, SNAP-25 composition further comprises an adjuvant, such as, e.g., polyethylene glycol (PEG), monomethoxypolyethylene glycol (mPEG), or polyvinyl alcohol (PVA).

[0052] In another embodiment, a method of producing .alpha.-SNAP-25 antibodies that bind an epitope comprising a carboxyl-terminus at the P.sub.1 residue from the BoNT/A cleavage site scissile bond from a SNAP-25 cleavage product comprises the steps (a) administering to an animal a composition comprising a SNAP-25 peptide having a carboxylated C-terminal glutamine linked to a flexible linker and a carrier peptide wherein the flexible linker intervenes between the SNAP-25 peptide and the carrier peptide; (b) collecting from the animal a sample containing an .alpha.-SNAP-25 antibody or .alpha.-SNAP-25 antibody-producing cell; and (c) isolating the .alpha.-SNAP-25 antibody from the sample. In an aspect of this embodiment, the .alpha.-SNAP-25 antibodies that bind an epitope comprising a carboxyl-terminus at the P1 residue from the BoNT/A cleavage site scissile bond from a SNAP-25 cleavage product is a polyclonal antibody. In another aspect of this embodiment, .alpha.-SNAP-25 antibodies that bind an epitope comprising a carboxyl-terminus at the P1 residue from the BoNT/A cleavage site scissile bond from a SNAP-25 cleavage product is a monoclonal antibody. In a further aspect of this embodiment, an .alpha.-SNAP-25 monoclonal antibody that binds an epitope comprising a carboxyl-terminus at the P1 residue from the BoNT/A cleavage site scissile bond from a SNAP-25 cleavage product produced in an IgG subtype. In other aspects of this embodiment, SNAP-25 composition further comprises an adjuvant, such as, e.g., polyethylene glycol (PEG), monomethoxypolyethylene glycol (mPEG), or polyvinyl alcohol (PVA).

[0053] Aspects of the present disclosure comprise, in part, an isolated .alpha.-SNAP-25 antibody that selectively binds to a SNAP-25 epitope having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond. As used herein, the term "isolated" refers to separating a molecule from its natural environment by the use of human intervention. As used herein, the term "antibody" refers to a molecule generated by an immune system that was made in response to a particular antigen that specifically binds to that antigen, and includes both naturally occurring antibodies and non-naturally occurring antibodies. As used herein, the term ".alpha.-SNAP-25" is synomonous with "anti-SNAP-25" and refers to an antibody that binds to a SNAP-25 antigen. For example, an antibody can be a polyclonal antibody, a monoclonal antibody, a dimer, a multimer, a multispecific antibody, a humanized antibody, a chimeric antibody, bi-functional antibody, a cell-associated antibody like an Ig receptor, a linear antibody, a diabody, or a minibody, so long as the fragment exhibits the desired biological activity, and single chain derivatives of the same. An antibody can be a full-length immunoglobulin molecule comprising the V.sub.H and V.sub.L domains, as well as a light chain constant domain (C.sub.L) and heavy chain constant domains, C.sub.H1, C.sub.H2 and C.sub.H3, or an immunologically active fragment of a full-length immunoglobulin molecule, such as, e.g., a Fab fragment, a F(ab').sub.2 fragment, a Fc fragment, a Fd fragment, a Fv fragment. An antibody can be derived from any vertebrate species (e.g., human, goat, horse, donkey, murine, rat, rabbit, or chicken), and can be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgA, IgD, IgE, IgG, and IgM) or subclass (IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2). For general disclosure on the structure of naturally occurring antibodies, non-naturally occurring antibodies, and antigenic compound-binding fragments thereof, see, e.g., Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994); Borrabeck, Antibody Engineering, 2d ed. (Oxford University Press 1995), each of which is hereby incorporated by reference in its entirety.

[0054] Naturally-occurring antibodies are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (V.sub.H) followed by a number of constant domains. Each light chain has a variable domain at one end (V.sub.L) and a constant domain at its other end. The constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light-chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains.

[0055] The complete antigen-recognition and antigen-binding site is contained within the variable domains of the antibody, i.e., the Fv fragment. This fragment includes a dimer of one heavy chain variable domain (V.sub.H) and one light chain variable domain (V.sub.L) in tight, non-covalent association. Each domain comprises four framework regions (FR), which largely adopting a .beta.-sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases form part of, the .beta.-sheet structure. Each hypervariable region comprises an amino acid sequence corresponding to a complementarity determining region (CDRs). Collectively, it the three-dimensional configuration of the six CDR regions that define an antigen-binding site on the surface of the V.sub.H-V.sub.L dimmer that confers antigen-binding specificity. See e.g., Cyrus Chothia, et al., Conformations of Immunoglobulin Hypervariable Regions, Nature 342(6252): 877-883 (1989); Elvin A. Kabat, et al Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991), each of which is incorporated by reference in its entirety. The constant domains of the antibody are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody dependent cellular cytotoxicity.

[0056] A target antigen generally has one or more binding sites, also called epitopes, which are recognized by the CDR-formed antigen-binding site. As used herein, an "epitope" is synonymous with "antigenic determinant" and refers to the site on a target antigen, such as, e.g., a peptide, polysaccharide or lipid-containing molecule, capable of specific binding to an immunoglobulin or T-cell receptor or otherwise interacting with a molecule. Each antibody that specifically binds to a different epitope has a different structure. Thus, one antigen may have more than one corresponding antibody.

[0057] Polyclonal antibodies refer to a heterogeneous population of antibody molecules that contain at least two species of antibody capable of binding to a particular antigen. By definition, a polyclonal antibody includes two different antibodies that bind to at least two different epitopes. As used herein, the term "monoclonal antibody" or "monoclonal antibodies" refer to a substantially homogeneous population of antibody molecules that contain only one species of antibody capable of binding a particular antigen i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. By definition, a monoclonal antibody binds to a single epitope. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibodies, each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present disclosure may be made by the hybridoma method first described by Kohler et al (1975) Nature 256:495, or may be made by recombinant DNA methods (see for example: U.S. Pat. Nos. 4,816,567; 5,807,715). The monoclonal antibodies may also be isolated from phage antibody libraries using the techniques described in Clackson et al (1991) Nature, 352:624-628; Marks et al (1991) J. Mol. Biol., 222:581-597; for example.

[0058] Thus, in an embodiment, an .alpha.-SNAP-25 antibody comprises a heavy chain variable domain (V.sub.H) and a light chain variable domain (V.sub.L) that selectively binds to a SNAP-25 having a carboxyl-terminus at the P1 residue of the BoNT/A cleavage site scissile bond. In an aspect of this embodiment, the heavy chain variable domain (V.sub.H) is SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 80, or SEQ ID NO: 82. In another aspect of this embodiment, the light chain variable domain (V.sub.L) is SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, or SEQ ID NO: 92.

[0059] In another embodiment, an .alpha.-SNAP-25 antibody comprises a heavy chain variable domain (V.sub.H) CDR1 region, a CDR2 region, a CDR3 region, or any combination thereof that selectively binds to a SNAP-25 having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond. In an aspect of this embodiment, the heavy chain variable domain (V.sub.H) CDR1 region is SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 118, SEQ ID NO: 119, or SEQ ID NO: 120. In another aspect of this embodiment, the heavy chain variable domain (V.sub.H) CDR2 region is SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 121, SEQ ID NO: 122, or SEQ ID NO: 123. In yet another aspect of this embodiment, the heavy chain variable domain (V.sub.H) CDR3 region is SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, or SEQ ID NO: 124.

[0060] In another embodiment, an .alpha.-SNAP-25 antibody comprises a light chain variable domain (V.sub.L) CDR1 region, a CDR2 region, a CDR3 region, or any combination thereof that selectively binds to a SNAP-25 having a carboxyl-terminus at the P1 residue of the BoNT/A cleavage site scissile bond. In an aspect of this embodiment, the light chain variable domain (V.sub.L) CDR1 region is SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, or SEQ ID NO: 129. In another aspect of this embodiment, the light chain variable domain (V.sub.L) CDR2 region is SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, or SEQ ID NO: 112. In yet another aspect of this embodiment, the light chain variable domain (V.sub.L) CDR3 region is SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, or SEQ ID NO: 117.

[0061] In yet another embodiment, an .alpha.-SNAP-25 antibody specifically binds an epitope comprising a SNAP-25 having a carboxyl-terminus at the P1 residue of the BoNT/A cleavage site scissile bond. In an aspect of this embodiment, the epitope comprises SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 147 or SEQ ID NO: 148. In an aspect of this embodiment, the epitope comprises SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, or SEQ ID NO: 44.

[0062] As discussed above, the sequence surrounding a BoNT/A cleavage site present in SNAP-25 is denoted P.sub.5-P.sub.4-P.sub.3-P.sub.2-P.sub.1-P.sub.1'-P.sub.2'-P.sub.3'-P.sub.- 4'-P.sub.5', with P.sub.1-P.sub.1' representing the scissile bond. Upon cleavage by BoNT/A, the resulting cleavage products produced comprise a fragment including the P.sub.5-P.sub.4-P.sub.3-P.sub.2-P.sub.1 sequence and a fragment including the P.sub.1'-P.sub.2'-P.sub.3'-P.sub.4'-P.sub.5'. As used herein, the term ".alpha.-SNAP-25 antibodies that bind an epitope comprising a carboxyl-terminus at the P.sub.1 residue from the BoNT/A cleavage site scissile bond from a SNAP-25 cleavage product" refers to .alpha.-SNAP-25 antibodies that selectively bind to any SNAP-25 cleavage product fragment comprising the P.sub.5-P.sub.4-P.sub.3-P.sub.2-P.sub.1 sequence, but not to any SNAP-25 cleavage product fragment comprising the P.sub.1'-P.sub.2'-P.sub.3'-P.sub.4'-P.sub.5' sequence or to any SNAP-25 having an intact P.sub.1-P.sub.1' scissile bond of a BoNT/A cleavage site. As used herein, the term ".alpha.-SNAP-25.sub.197 antibody" refers to an antibody that selectively binds to a SNAP-25 having a carboxyl-terminus P.sub.1 residue that corresponds to glutamine 197 of SEQ ID NO: 5. As used herein, the term ".alpha.-SNAP-25.sub.204 antibody" refers to an antibody that selectively binds to a SNAP-25 having a carboxyl-terminus P.sub.1 residue that corresponds to lysine 204 of SEQ ID NO: 16.

[0063] As used herein, the term "selectively" refers to having a unique effect or influence or reacting in only one way or with only one thing. As used herein, the term "selectively binds," when made in reference to an antibody, refers to the discriminatory binding of the antibody to the indicated target epitope such that the antibody does not substantially cross react with non-target epitopes. The minimal size of a peptide epitope, as defined herein, is about five amino acids, and a peptide epitope typically comprises at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, or at least 20 amino acids. A peptide epitope may be discontinuous, i.e., it comprises amino acid residues that are not adjacent in the primary structure of the peptide but are brought together into an epitope by way of the secondary, tertiary, or quaternary structure of the peptide. Furthermore, it is also noted that an epitope might comprise a portion of a molecule other than an amino acid sequence, such as, e.g., a carbohydrate moiety, a lipid moiety like lipoproteins or glycolipids, or a chemically-modified amino acid moiety like a phosphorylated amino acid. In aspects of this embodiment, an .alpha.-SNAP-25 antibody that selectively binds to a SNAP-25 epitope having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond can selectively bind a SNAP-25 epitope having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond comprising at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, or at least 20 amino acids. In other aspects of this embodiment, an .alpha.-SNAP-25 antibody that selectively binds to a SNAP-25 epitope having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond can selectively bind a SNAP-25 epitope having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond comprising at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 15, or at most 20 amino acids.

[0064] Selective binding includes binding properties such as, e.g., binding affinity, binding specificity, and binding avidity. See David J. King, Applications and Engineering of Monoclonal Antibodies, pp. 240 (1998). Binding affinity refers to the length of time the antibody resides at its epitope binding site, and can be viewed as the strength with which an antibody binds its epitope. Binding affinity can be described an antibody's equilibrium dissociation constant (KD), which is defined as the ratio Kd/Ka at equillibrium. Where Ka is the antibody's association rate constant and kd is the antibody's dissociation rate constant. Binding affinity is determined by both the association and the dissociation and alone neither high association or low dissociation can ensure high affinity. The association rate constant (Ka), or on-rate constant (Kon), measures the number of binding events per unit time, or the propensity of the antibody and the antigen to associate reversibly into its antibody-antigen complex. The association rate constant is expressed in M.sup.-1 s.sup.-1, and is symbolized as follows: [Ab].times.[Ag].times.Kon. The larger the association rate constant, the more rapidly the antibody binds to its antigen, or the higher the binding affinity between antibody and antigen. The dissociation rate constant (Kd), or off-rate constant (Koff), measures the number of dissociation events per unit time propensity of an antibody-antigen complex to separate (dissociate) reversibly into its component molecules, namely the antibody and the antigen. The dissociation rate constant is expressed in s.sup.-1, and is symbolized as follows: [Ab+Ag].times.Koff. The smaller the dissociation rate constant, the more tightly bound the antibody is to its antigen, or the higher the binding affinity between antibody and antigen. The equilibrium dissociation constant (KD) measures the rate at which new antibody-antigen complexes formed equals the rate at which antibody-antigen complexes dissociate at equilibrium. The equilibrium dissociation constant is expressed in M, and is defined as Koff/Kon=[Ab].times.[Ag]/[Ab+Ag], where [Ab] is the molar concentration of the antibody, [Ag] is the molar concentration of the antigen, and [Ab+Ag] is the of molar concentration of the antibody-antigen complex, where all concentrations are of such components when the system is at equilibrium. The smaller the equilibrium dissociation constant, the more tightly bound the antibody is to its antigen, or the higher the binding affinity between antibody and antigen.

[0065] Thus, in an embodiment, the binding affinity of an .alpha.-SNAP-25 antibody that selectively binds to a SNAP-25 epitope having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond can have an association rate constant of, e.g., less than 1.times.10.sup.5 M.sup.-1 s.sup.-1, less than 1.times.10.sup.6 M.sup.-1 s.sup.-1, less than 1.times.10.sup.7 M.sup.-1 s.sup.-1, or less than 1.times.10.sup.8 M.sup.-1 s.sup.-1. In another embodiment, the binding affinity of an .alpha.-SNAP-25 antibody that selectively binds to a SNAP-25 epitope having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond can have an association rate constant of, e.g., more than 1.times.10.sup.5 M.sup.-1 s.sup.-1, more than 1.times.10.sup.6 M.sup.-1 s.sup.-1, more than 1.times.10.sup.7 M.sup.-1 s.sup.-1, or more than 1.times.10.sup.8 M.sup.-1 s.sup.-1. In other aspects, the binding affinity of an .alpha.-SNAP-25 antibody that selectively binds to a SNAP-25 epitope having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond can have an association rate constant between 1.times.10.sup.5 M.sup.-1 s.sup.-1 to 1.times.10.sup.8 M.sup.-1 s.sup.-1, 1.times.10.sup.6 M.sup.-1 s.sup.-1 to 1.times.10.sup.8 M.sup.-1, 1.times.10.sup.5 M.sup.-1 s.sup.-1 to 1.times.10.sup.7 M.sup.-1 s.sup.-1, or 1.times.10.sup.6 Mm.sup.-1 s.sup.-1 to 1.times.10.sup.7 M.sup.-1 s.sup.-1.

[0066] In another embodiment, the binding affinity of an .alpha.-SNAP-25 antibody that selectively binds to a SNAP-25 epitope having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond can have a disassociation rate constant of less than 1.times.10.sup.-3 s.sup.-1, less than 1.times.10.sup.-4 s.sup.-1, or less than 1.times.10.sup.-5 s.sup.-1. In other aspects of this embodiment, the binding affinity of an .alpha.-SNAP-25 antibody that selectively binds to a SNAP-25 epitope having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond can have a disassociation rate constant of, e.g., less than 1.0.times.10.sup.-4 s.sup.-1, less than 2.0.times.10.sup.-4 s.sup.-1, less than 3.0.times.10.sup.-4 s.sup.-1, less than 4.0.times.10.sup.-4 s.sup.-1, less than 5.0.times.10.sup.-4 s.sup.-1, less than 6.0.times.10.sup.-4 s.sup.-1, less than 7.0.times.10.sup.-4 s.sup.-1, less than 8.0.times.10.sup.-4 s.sup.-1, or less than 9.0.times.10.sup.-4 s.sup.-1. In another embodiment, the binding affinity of an .alpha.-SNAP-25 antibody that selectively binds to a SNAP-25 epitope having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond can have a disassociation rate constant of, e.g., more than 1.times.10.sup.-3 s.sup.-1, more than 1.times.10.sup.-4 s.sup.-1, or more than 1.times.10.sup.-5 s.sup.-1. In other aspects of this embodiment, the binding affinity of an .alpha.-SNAP-25 antibody that selectively binds to a SNAP-25 epitope having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond can have a disassociation rate constant of, e.g., more than 1.0.times.10.sup.-4 s.sup.-1, more than 2.0.times.10.sup.-4 s.sup.-1, more than 3.0.times.10.sup.-4 s.sup.-1, more than 4.0.times.10.sup.-4 s.sup.-1, more than 5.0.times.10.sup.-4 s.sup.-1, more than 6.0.times.10.sup.-4 s.sup.-1, more than 7.0.times.10.sup.-4 s.sup.-1, more than 8.0.times.10.sup.-4 s.sup.-1, or more than 9.0.times.10.sup.-4 s.sup.-1.

[0067] In another embodiment, the binding affinity of an .alpha.-SNAP-25 antibody that selectively binds to a SNAP-25 epitope having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond can have an equilibrium disassociation constant of less than 0.500 nM. In aspects of this embodiment, the binding affinity of an .alpha.-SNAP-25 antibody that selectively binds to a SNAP-25 epitope having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond can have an equilibrium disassociation constant of, e.g., less than 0.500 nM, less than 0.450 nM, less than 0.400 nM, less than 0.350 nM, less than 0.300 nM, less than 0.250 nM, less than 0.200 nM, less than 0.150 nM, less than 0.100 nM, or less than 0.050 nM. In another embodiment, the binding affinity of an .alpha.-SNAP-25 antibody that selectively binds to a SNAP-25 epitope having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond can have an equilibrium disassociation constant of more than 0.500 nM. In aspects of this embodiment, the binding affinity of an .alpha.-SNAP-25 antibody that selectively binds to a SNAP-25 epitope having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond can have an equilibrium disassociation constant of, e.g., more than 0.500 nM, more than 0.450 nM, more than 0.400 nM, more than 0.350 nM, more than 0.300 nM, more than 0.250 nM, more than 0.200 nM, more than 0.150 nM, more than 0.100 nM, or more than 0.050 nM.

[0068] In yet another embodiment, the binding affinity of an .alpha.-SNAP-25 antibody that selectively binds to a SNAP-25 epitope having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond can have an association rate constant of for the intact SNAP-25 of, e.g., less than 1.times.10.sup.0 M.sup.-1 s.sup.-1, less than 1.times.10.sup.1 M.sup.-1 s.sup.-1, less than 1.times.10.sup.2 M.sup.-1 s.sup.-1, less than 1.times.10.sup.3 M.sup.-1 s.sup.-1, or less than 1.times.10.sup.4 M.sup.-1 s.sup.-1. In another embodiment, the binding affinity of an .alpha.-SNAP-25 antibody that selectively binds to a SNAP-25 epitope having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond can have an association rate constant of for the intact SNAP-25 of, e.g., at most 1.times.10.sup.0 M.sup.-1 s.sup.-1, at most 1.times.10.sup.1 M.sup.-1 s.sup.-1, at most 1.times.10.sup.2 M.sup.-1 s.sup.-1, at most 1.times.10.sup.3 M.sup.-1 s.sup.-1, or at most 1.times.10.sup.4 M.sup.-1 s.sup.-1.

[0069] Binding specificity is the ability of an antibody to discriminate between a molecule containing its epitope and a molecule that does not contain that epitope. One way to measure binding specificity is to compare the Kon association rate of the antibody for a molecule containing its epitope relative to the Kon association rate of the antibody for a molecule that does not contain that epitope. For example, comparing the association rate constant (Ka) of an .alpha.-SNAP-25 antibody for a SNAP-25 epitope having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond relative to a SNAP-25 not comprising that epitope, such as, e.g., a SNAP-25 epitope lacking a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond or a SNAP-25 epitope having an intact P.sub.1-P.sub.1' scissile bond of a BoNT/A cleavage site. In aspects of this embodiment, an .alpha.-SNAP-25 antibody that selectively binds to a SNAP-25 epitope having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond has an association rate constant (Ka) for a SNAP-25 not comprising its epitope(s) of, e.g., less than 1.times.10.sup.0 M.sup.-1 s.sup.-1, less than 1.times.10.sup.1 M.sup.-1 s.sup.-1, less than 1.times.10.sup.2 M.sup.-1 s.sup.-1, less than 1.times.10.sup.3 M.sup.-1 s.sup.-1 or less than 1.times.10.sup.4 M.sup.-1 s.sup.-1. In other aspects of this embodiment, an .alpha.-SNAP-25 antibody that selectively binds to a SNAP-25 epitope having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond has an association rate constant (Ka) for a SNAP-25 not comprising its epitope(s) of, e.g., at most 1.times.10.sup.0 M.sup.-1 s.sup.-1, at most 1.times.10.sup.1 M.sup.-1 s.sup.-1, at most 1.times.10.sup.2 M.sup.-1 s.sup.-1, at most 1.times.10.sup.3 M.sup.-1 s.sup.-1 or at most 1.times.10.sup.4 M.sup.-1 s.sup.-1.

[0070] In yet aspects of this embodiment, an .alpha.-SNAP-25 antibody that selectively binds to a SNAP-25 epitope having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond has an association rate constant (Ka) for its epitope relative to a SNAP-25 not comprising that epitope of, e.g., at least 2-fold more, at least 3-fold more, at least 4-fold more, at least 5-fold more, at least 6-fold more, at least 7-fold more, at least 8-fold more, or at least 9-fold more. In further aspects of this embodiment, an .alpha.-SNAP-25 antibody that selectively binds to a SNAP-25 epitope having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond has an association rate constant (Ka) for its epitope relative to a SNAP-25 not comprising that epitope of, e.g., at least 10-fold more, at least 100-fold more, at least 1,000-fold more or at least 10,000-fold more. In yet other aspects of this embodiment, an .alpha.-SNAP-25 antibody that selectively binds to a SNAP-25 epitope having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond has an association rate constant (Ka) for its epitope relative to a SNAP-25 not comprising that epitope of, e.g., at most 1-fold more, at most 2-fold more, at most 3-fold more, at most 4-fold more, at most 5-fold more, at most 6-fold more, at most 7-fold more, at most 8-fold more, or at most 9-fold more. In yet other aspects of this embodiment, an .alpha.-SNAP-25 antibody that selectively binds to a SNAP-25 epitope having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond has an association rate constant (Ka) for its epitope relative to a SNAP-25 not comprising that epitope of, e.g., at most 10-fold more, at most 100-fold more, at most 1,000-fold more or at most 10,000-fold more.

[0071] The binding specificity of an .alpha.-SNAP-25 antibody that selectively binds to a SNAP-25 epitope having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond can also be characterized as a ratio that such an .alpha.-SNAP-25 antibody can discriminate its SNAP-25 epitope relative to a SNAP-25 not comprising that epitope, such as, e.g., a SNAP-25 epitope lacking a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond or a SNAP-25 epitope having an intact P.sub.1-P.sub.1' scissile bond of a BoNT/A cleavage site. In aspects of this embodiment, an .alpha.-SNAP-25 antibody that selectively binds to a SNAP-25 epitope having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond has a binding specificity ratio for its SNAP-25 epitope relative to a SNAP-25 not comprising that epitope of, e.g., at least 2:1, at least 3:1, at least 4:1, at least 5:1, at least 64:1, at least 7:1, at least 8:1, at least 9:1, at least 10:1, at least 15:1, at least 20:1, at least 25:1, at least 30:1, at least 35:1, or at least 40:1. In yet other aspects of this embodiment, an .alpha.-SNAP-25 antibody that selectively binds to a SNAP-25 epitope having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond has a binding specificity ratio for its SNAP-25 epitope relative to a SNAP-25 lacking a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond of, e.g., at least 2:1, at least 3:1, at least 4:1, at least 5:1, at least 6:1, at least 7:1, at least 8:1, at least 9:1, at least 10:1, at least 15:1, at least 20:1, at least 25:1, at least 30:1, at least 35:1, or at least 40:1. In still other aspects of this embodiment, an .alpha.-SNAP-25 antibody that selectively binds to a SNAP-25 epitope having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond has a binding specificity ratio for its SNAP-25 epitope relative to a SNAP-25 having an intake P.sub.1-P.sub.1' scissile bond of a BoNT/A cleavage site of, e.g., at least 2:1, at least 3:1, at least 4:1, at least 5:1, at least 64:1, at least 7:1, at least 8:1, at least 9:1, at least 10:1, at least 15:1, at least 20:1, at least 25:1, at least 30:1, at least 35:1, or at least 40:1.

[0072] Binding avidity, also known as functional affinity, refers to the sum total of the functional binding strength between a multivalent antibody and its antigen. Antibody molecules can have more than one binding site (e.g., 2 for IgG, 10 for IgM), and many antigens contain more than one antigenic site. While binding avidity of an antibody depends on the binding affinities of the individual antibody binding sites, binding avidity is greater than the binding affinity as all the antibody-antigen interactions must be broken simultaneously for the antibody to dissociate completely. It is envisioned that an .alpha.-SNAP-25 antibody that selectively binds to a SNAP-25 epitope having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond can selectively bind to any and all epitopes for that antibody.

[0073] Thus, in an embodiment, an .alpha.-SNAP-25 antibody is an .alpha.-SNAP-25 antibody that selectively binds to a SNAP-25 epitope having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond. In aspects of this embodiment, an .alpha.-SNAP-25 antibody is an .alpha.-SNAP-25 antibody that selectively binds to a SNAP-25 epitope having a carboxyl-terminus glutamine or an .alpha.-SNAP-25 antibody that selectively binds to a SNAP-25 epitope having a carboxyl-terminus lysine. In other aspects of this embodiment, an .alpha.-SNAP-25 antibody is an .alpha.-SNAP-25 antibody that selectively binds to a SNAP-25 epitope having a carboxyl-terminus P.sub.1 residue that corresponds to glutamine 197 of SEQ ID NO: 5 or an .alpha.-SNAP-25 antibody that selectively binds to a SNAP-25 epitope having a carboxyl-terminus P.sub.1 residue that corresponds to lysine 204 of SEQ ID NO: 16. In still other aspects of this embodiment, an .alpha.-SNAP-25 antibody is an .alpha.-SNAP-25 antibody that selectively binds to a SNAP-25 epitope having a carboxyl-terminal amino acid sequence of SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 147 or SEQ ID NO: 148.

[0074] Aspects of the present disclosure comprise, in part, an immuno-based method of detecting BoNT/A activity. The immuno-based methods disclosed in the present specification can be evaluated by several parameters including, e.g., accuracy, precision, limit of detection (LOD), limits of quantitation (LOQ), linear range, specificity, selectivity, linearity, ruggedness, and system suitability. The accuracy of a method is the measure of exactness of an analytical method, or the closeness of agreement between the measured value and the value that is accepted as a conventional true value or an accepted reference value. The precision of a method is the degree of agreement among individual test results, when the procedure is applied repeatedly to multiple samplings of a homogeneous sample. As such, precision evaluates 1) within assay variability; 2) within-day variability (repeatability); and 3) between-day variability (intermediate precision); and 4) between-lab variability (reproducibility). Coefficient of variation (CV %) is a quantitative measure of precision expressed relative to the observed or theoretical mean value.

[0075] An immuno-based method disclosed in the present specification must be able to detect, over background, the presence of an .alpha.-SNAP-25 antibody-antigen complex comprising a SNAP-25 having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond. The limit of detection (LOD) of a method refers to the concentration of analyte which gives rise to a signal that is significantly different from the negative control or blank and represents the lowest concentration of analyte that can be distinguished from background.

[0076] Thus, in an embodiment, the immuno-based method disclosed in the present specification can detect the LOD of BoNT/A at an amount that is significantly different from a negative control or blank. In aspect of this embodiment, the immuno-based method disclosed in the present specification has an LOD of, e.g., 10 ng or less, 9 ng or less, 8 ng or less, 7 ng or less, 6 ng or less, 5 ng or less, 4 ng or less, 3 ng or less, 2 ng or less, 1 ng or less of a BoNT/A. In still other aspects of this embodiment, the immuno-based method disclosed in the present specification has an LOD of, e.g., 900 pg or less, 800 pg or less, 700 pg or less, 600 pg or less, 500 pg or less, 400 pg or less, 300 pg or less, 200 pg or less, 100 pg or less of a BoNT/A. In further aspects of this embodiment, the immuno-based method disclosed in the present specification has an LOD of, e.g., 90 pg or less, 80 pg or less, 70 pg or less, 60 pg or less, 50 pg or less, 40 pg or less, 30 pg or less, 20 pg or less, 10 pg or less of a BoNT/A. In other aspects of this embodiment, the immuno-based method disclosed in the present specification has an LOD of, e.g., 9 pg or less, 8 pg or less, 7 pg or less, 6 pg or less, 5 pg or less, 4 pg or less, 3 pg or less, 2 pg or less, 1 pg or less of a BoNT/A. In yet other aspects of this embodiment, the immuno-based method disclosed in the present specification has an LOD of, e.g., 0.9 pg or less, 0.8 pg or less, 0.7 pg or less, 0.6 pg or less, 0.5 pg or less, 0.4 pg or less, 0.3 pg or less, 0.2 pg or less, 0.1 pg or less of a BoNT/A.

[0077] In another aspect of this embodiment, the immuno-based method disclosed in the present specification has an LOD of, e.g., 10 nM or less or less, 9 nM or less, 8 nM or less, 7 nM or less, 6 nM or less, 5 nM or less, 4 nM or less, 3 nM or less, 2 nM or less, or 1 nM or less of a BoNT/A. In other aspects of this embodiment, the immuno-based method disclosed in the present specification has an LOD of, e.g., 900 pM or less, 800 pM or less, 700 pM or less, 600 pM or less, 500 pM or less, 400 pM or less, 300 pM or less, 200 pM or less, or 100 pM or less of a BoNT/A. In other aspects of this embodiment, the immuno-based method disclosed in the present specification has an LOD of, e.g., 100 pM or less, 90 pM or less, 80 pM or less, 70 pM or less, 60 pM or less, 50 pM or less, 40 pM or less, 30 pM or less, 20 pM or less, or 10 pM or less of a BoNT/A. In yet other aspects of this embodiment, the immuno-based method disclosed in the present specification has an LOD of, e.g., 10 pM or less of a BoNT/A, 9 pM or less, 8 pM or less, 7 pM or less, 6 pM or less, 5 pM or less, 4 pM or less, 3 pM or less, 2 pM or less, or 1 pM or less of a BoNT/A. In still other aspects of this embodiment, the immuno-based method disclosed in the present specification has an LOD of, e.g., 1000 fM or less, 900 fM or less, 800 fM or less, 700 fM or less, 600 fM or less, 500 fM or less, 400 fM or less, 300 fM or less, 200 fM or less, or 100 fM or less of a BoNT/A. In still other aspects of this embodiment, the immuno-based method disclosed in the present specification has an LOD of, e.g., 100 fM or less, 90 fM or less, 80 fM or less, 70 fM or less, 60 fM or less, 50 fM or less, 40 fM or less, 30 fM or less, 20 fM or less, or 10 fM or less of a BoNT/A. In still other aspects of this embodiment, the immuno-based method disclosed in the present specification has an LOD of, e.g., 10 fM or less, 9 fM or less, 8 fM or less, 7 fM or less, 6 fM or less, 5 fM or less, 4 fM or less, 3 fM or less, 2 fM or less, or 1 fM or less of a botulinum neurotoxin A.

[0078] The limits of quantitation (LOQ) are the lowest and the highest concentrations of analyte in a sample or specimen that can be measured with an acceptable level of accuracy and precision. The lower limit of quantitation refers to the lowest dose that a detection method can measure consistently from the background. The upper limit of quantitation is the highest dose that a detection method can measure consistently before saturation of the signal occurs. The linear range of the method is the area between the lower and the upper limits of quantitation. The linear range is calculated by subtracting lower limit of quantitation from the upper limit of quantitation. As used herein, the term "signal to noise ratio for the lower asymptote" refers to the signal detected in the method at the lower limit of detection divided by the background signal. As used herein, the term "signal to noise ratio for the upper asymptote" refers to the signal detected in the method at the upper limit of detection divided by the background signal.

[0079] Thus, in an embodiment, the immuno-based method disclosed in the present specification can detect the LOQ of BoNT/A at an amount that is significantly different from a negative control or blank. In aspect of this embodiment, the immuno-based method disclosed in the present specification has an LOQ of, e.g., 10 ng or less, 9 ng or less, 8 ng or less, 7 ng or less, 6 ng or less, 5 ng or less, 4 ng or less, 3 ng or less, 2 ng or less, 1 ng or less of a BoNT/A. In still other aspects of this embodiment, the immuno-based method disclosed in the present specification has an LOQ of, e.g., 900 pg or less, 800 pg or less, 700 pg or less, 600 pg or less, 500 pg or less, 400 pg or less, 300 pg or less, 200 pg or less, 100 pg or less of a BoNT/A. In further aspects of this embodiment, the immuno-based method disclosed in the present specification has an LOQ of, e.g., 90 pg or less, 80 pg or less, 70 pg or less, 60 pg or less, 50 pg or less, 40 pg or less, 30 pg or less, 20 pg or less, 10 pg or less of a BoNT/A. In other aspects of this embodiment, the immuno-based method disclosed in the present specification has an LOQ of, e.g., 9 pg or less, 8 pg or less, 7 pg or less, 6 pg or less, 5 pg or less, 4 pg or less, 3 pg or less, 2 pg or less, 1 pg or less of a BoNT/A. In yet other aspects of this embodiment, the immuno-based method disclosed in the present specification has an LOQ of, e.g., 0.9 pg or less, 0.8 pg or less, 0.7 pg or less, 0.6 pg or less, 0.5 pg or less, 0.4 pg or less, 0.3 pg or less, 0.2 pg or less, 0.1 pg or less of a BoNT/A.

[0080] In another aspect of this embodiment, the immuno-based method disclosed in the present specification has an LOQ of, e.g., 10 nM or less, 9 nM or less, 8 nM or less, 7 nM or less, 6 nM or less, 5 nM or less, 4 nM or less, 3 nM or less, 2 nM or less, or 1 nM or less of a BoNT/A. In other aspects of this embodiment, the immuno-based method disclosed in the present specification has an LOQ of, e.g., 900 pM or less, 800 pM or less, 700 pM or less, 600 pM or less, 500 pM or less, 400 pM or less, 300 pM or less, 200 pM or less, or 100 pM or less of a BoNT/A. In other aspects of this embodiment, the immuno-based method disclosed in the present specification has an LOQ of, e.g., 100 pM or less, 90 pM or less, 80 pM or less, 70 pM or less, 60 pM or less, 50 pM or less, 40 pM or less, 30 pM or less, 20 pM or less, or 10 pM or less of a BoNT/A. In yet other aspects of this embodiment, the immuno-based method disclosed in the present specification has an LOQ of, e.g., 10 pM or less of a BoNT/A, 9 pM or less, 8 pM or less, 7 pM or less, 6 pM or less, 5 pM or less, 4 pM or less, 3 pM or less, 2 pM or less, or 1 pM or less of a BoNT/A. In still other aspects of this embodiment, the immuno-based method disclosed in the present specification has an LOQ of, e.g., 1000 fM or less, 900 fM or less, 800 fM or less, 700 fM or less, 600 fM or less, 500 fM or less, 400 fM or less, 300 fM or less, 200 fM or less, or 100 fM or less of a BoNT/A. In still other aspects of this embodiment, the immuno-based method disclosed in the present specification has an LOQ of, e.g., 100 fM or less, 90 fM or less, 80 fM or less, 70 fM or less, 60 fM or less, 50 fM or less, 40 fM or less, 30 fM or less, 20 fM or less, or 10 fM or less of a BoNT/A. In still other aspects of this embodiment, the immuno-based method disclosed in the present specification has an LOQ of, e.g., 10 fM or less, 9 fM or less, 8 fM or less, 7 fM or less, 6 fM or less, 5 fM or less, 4 fM or less, 3 fM or less, 2 fM or less, or 1 fM or less of a BoNT/A.

[0081] An immuno-based assay useful to practice aspect of the disclosed methods must have a precision of no more than 50%. In aspects of this embodiment, an immuno-based assay has a precision of no more than 50%, no more than 40%, no more than 30%, or no more than 20%. In other aspects of this embodiment, an immuno-based assay has a precision of nor more than 15%, no more than 10%, or no more than 5%. In other aspects of this embodiment, an immuno-based assay has a precision of nor more than 4%, no more than 3%, no more than 2%, or no more than 1%.

[0082] An immuno-based assay useful to practice aspect of the disclosed methods must have an accuracy of at least 50%. In aspects of this embodiment, an immuno-based assay has an accuracy of at least 50%, at least 60%, at least 70%, or at least 80%. In other aspects of this embodiment, an immuno-based assay has an accuracy of at least 85%, at least 90%, or at least 95%. In other aspects of this embodiment, an immuno-based assay has an accuracy of at least 96%, at least 97%, at least 98%, or at least 99%.

[0083] An immuno-based method disclosed in the present specification must have a signal to noise ratio for the lower asymptote that is statistically significant and a signal to noise ratio for the upper asymptote that is statistically significant. In aspects of this embodiment, an immuno-based method disclosed in the present specification has a signal to noise ratio for the lower asymptote of, e.g., at least 3:1, at least 4:1, at least 5:1, at least 6:1, at least 7:1, at least 8:1, at least 9:1, at least 10:1, at least 15:1 or at least 20:1. In other aspects of this embodiment, an immuno-based method has a signal to noise ratio for the upper asymptote of, e.g., at least 10:1, at least 15:1, at least 20:1, at least 25:1, at least 30:1, at least 35:1, at least 40:1, at least 45:1, at least 50:1, at least 60:1, at least 70:1, at least 80:1, at least 90:1, or at least 100:1, at least 150:1, at least 200:1, at least 250:1, at least 300:1, at least 350:1, at least 400:1, at least 450:1, at least 500:1, at least 550:1, or at least 600:1.

[0084] The specificity of a method defines the ability of the method to measure the analyte of interest to the exclusion of other relevant components, such as, e.g., partially-active or inactive analyte. The selectivity of a method describes the ability of an analytical method to differentiate various substances in a sample. The linearity of a method is its ability to elicit results that are directly, or by a well defined mathematical transformation, proportional to the concentration of analyte in the sample. Thus in an embodiment, an immuno-based method disclosed in the present specification can distinguish a fully-active BoNT/A from a partially-active BoNT/A having, e.g., 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, or 10% or less the activity of a fully-active BoNT/A.

[0085] The ruggedness of the method is the reproducibility of the test results obtained for identical samples under normal (but variable) test conditions. Robustness of a procedure is a measure of its capacity to remain unaffected by small but deliberate variations in the method parameters and provides an indication of its reliability in normal usage. Thus, whereas ruggedness evaluates unavoidable changes, robustness evaluates deliberate changes. Typical parameters evaluated by ruggedness and robustness include the effects of freeze/thaw, incubation times, incubation temperature, longevity of reagent, sample preparation, sample storage, cell passage number, lots of toxin, variability between purifications, and variability between nicking reactions. Robustness parameters for cell-based assays include the cell bank (beginning, middle and end of freeze), cell passage level, cell seeding density, cell stock density (how many days in culture), cell age in flask (waiting time to seeding), incubation time, different plates, excessive amounts of serum, and source of reagents. The system suitability of the method is the determination of assay performance, including the performance of reagents and instruments, over time by analysis of a reference standard. System suitability is stressed in FDA guidance referring to the fact that equipment, electronics, assay performance, and samples to be analyzed, constitute an integrated system. System suitability can be evaluated by testing for parallelism, which is when plotting the log dose versus the response, serial dilutions of the reference and serial dilutions of the samples should give rise to parallel curves.

[0086] Aspects of the present disclosure comprise, in part, a cell from an established cell line. As used herein, the term "cell" refers to any eukaryotic cell susceptible to BoNT/A intoxication by a BoNT/A or any eukaryotic cell that can uptake a BoNT/A. The term cell encompasses cells from a variety of organisms, such as, e.g., murine, rat, porcine, bovine, equine, primate and human cells; from a variety of cell types such as, e.g., neuronal and non-neuronal; and can be isolated from or part of a heterogeneous cell population, tissue or organism. As used herein, the term "established cell line" is synonymous with "immortal cell line," or "transformed cell line" and refers to a cell culture of cells selected for indefinite propagation from a cell population derived from an organism, tissue, or organ source. By definition, an established cell line excludes a cell culture of primary cells. As used herein, the term "primary cells" are cells harvested directly from fresh tissues or organs and do not have the potential to propagate indefinitely. An established cell line can comprise a heterogeneous population of cells or a uniform population of cells. An established cell line derived from a single cell is referred to as a clonal cell line. An established cell line can be one whose cells endogenously express all component necessary for the cells to undergo the overall cellular mechanism whereby a BoNT/A proteolytically cleaves a SNAP-25 substrate and encompasses the binding of a BoNT/A to a BoNT/A receptor, the internalization of the neurotoxin/receptor complex, the translocation of the BoNT/A light chain from an intracellular vesicle into the cytoplasm and the proteolytic cleavage of a SNAP-25. Alternatively, an established cell line can be one whose cells have had introduced from an exogenous source at least one component necessary for the cells to undergo the overall cellular mechanism whereby a BoNT/A proteolytically cleaves a SNAP-25 substrate and encompasses the binding of a BoNT/A to a BoNT/A receptor, the internalization of the neurotoxin/receptor complex, the translocation of the BoNT/A light chain from an intracellular vesicle into the cytoplasm and the proteolytic cleavage of a SNAP-25. Also refereed to as a genetically-engineered cell line, cells from such an established cell line may, e.g., express an exogenous FGFR2, an exogenous FGFR3, an exogenous SV2, an exogenous SNAP-25, or any combination thereof.

[0087] Aspects of the present disclosure comprise, in part, a cell from an established cell line susceptible to BoNT/A intoxication. As used herein, the terms "cell(s) susceptible to BoNT/A intoxication," "cell(s) susceptible to BoNT/A intoxication by a BoNT/A," or "cell(s) from an established cell line susceptible to BoNT/A intoxication by a BoNT/A" refer to cell(s) that can undergo the overall cellular mechanism whereby a BoNT/A proteolytically cleaves a SNAP-25 substrate and encompasses the binding of a BoNT/A to a BoNT/A receptor, the internalization of the neurotoxin/receptor complex, the translocation of the BoNT/A light chain from an intracellular vesicle into the cytoplasm and the proteolytic cleavage of a SNAP-25. By definition, cell(s) susceptible to of BoNT/A intoxication must express, or be engineered to express, at least one BoNT/A receptor and at least one SNAP-25 substrate. As used herein, the terms "cell(s) that can uptake BoNT/A" or "cell(s) comprising an established cell line that can uptake BoNT/A" refer to cells that can undergo the overall cellular mechanism whereby a BoNT/A proteolytically cleaves a SNAP-25 substrate and encompasses the binding of a BoNT/A to a BoNT/A receptor, the internalization of the neurotoxin/receptor complex, the translocation of the BoNT/A light chain from an intracellular vesicle into the cytoplasm and the proteolytic cleavage of a SNAP-25. By definition, cell(s) that can uptake BoNT/A must express, or be engineered to express, at least one BoNT/A receptor and at least one SNAP-25 substrate.

[0088] Thus in an embodiment, cells from an established cell line are susceptible to BoNT/A intoxication. In aspects of this embodiment, cells from an established cell line are susceptible to BoNT/A intoxication by, e.g., about 500 pM or less, about 400 pM or less, about 300 pM or less, about 200 pM or less, or about 100 pM or less of a BoNT/A. In other aspects of this embodiment, cells from an established cell line are susceptible to BoNT/A intoxication by, e.g., about 90 pM or less, about 80 pM or less, about 70 pM or less, about 60 pM or less, about 50 pM or less, about 40 pM or less, about 30 pM or less, about 20 pM or less A, or about 10 pM or less of a BoNT/A. In still other aspects, cells from an established cell line are susceptible to BoNT/A intoxication by, e.g., about 9 pM or less, about 8 pM or less, about 7 pM or less, about 6 pM or less, about 5 pM or less, about 4 pM or less, about 3 pM or less, about 2 pM or less, or about 1 pM or less of a BoNT/A. In yet other aspects, cells from an established cell line are susceptible to BoNT/A intoxication by, e.g., about 0.9 pM or less, about 0.8 pM or less, about 0.7 pM or less, about 0.6 pM or less, about 0.5 pM or less, about 0.4 pM or less, about 0.3 pM or less, about 0.2 pM, or about 0.1 pM or less of a BoNT/A. As used herein, the term "about" when qualifying a value of a stated item, number, percentage, or term refers to a range of plus or minus ten percent of the value of the stated item, percentage, parameter, or term.

[0089] In another embodiment, cells comprising an established cell line can uptake a BoNT/A. In aspects of this embodiment, cells comprising an established cell line can uptake, e.g., about 500 pM or less, about 400 pM or less, about 300 pM or less, about 200 pM or less, or about 100 pM or less of a BoNT/A. In other aspects of this embodiment, cells comprising an established cell line possess the ability to uptake about 90 pM or less, about 80 pM or less, about 70 pM or less, about 60 pM or less, about 50 pM or less, about 40 pM or less, about 30 pM or less, about 20 pM or less, or about 10 pM or less of a BoNT/A. In still other aspects, cells comprising an established cell line possess the ability to uptake about 9 pM or less, about 8 pM or less, about 7 pM or less, about 6 pM or less, about 5 pM or less, about 4 pM or less, about 3 pM or less, about 2 pM or less, or about 1 pM or less of a BoNT/A. In yet other aspects, cells comprising an established cell line possess the ability to uptake about 0.9 pM or less, about 0.8 pM or less, about 0.7 pM or less, about 0.6 pM or less, about 0.5 pM or less, about 0.4 pM or less, about 0.3 pM or less, about 0.2 pM or less, or about 0.1 pM or less of a BoNT/A.

[0090] Aspects of the present disclosure comprise, in part, a BoNT/A. As used herein, the term "BoNT/A" is synonymous with "botulinum neurotoxin serotype A" or "botulinum neurotoxin type A" and refers to both a naturally-occurring BoNT/A or a non-naturally occurring BoNT/As thereof, and includes BoNT/A complex comprising the about 150 kDa BoNT/A neurotoxin and associated non-toxin associated proteins (NAPs), as well as the about 150 kDa BoNT/A neurotoxin alone. Non-limiting examples of BoNT/A complexes include, e.g., the 900-kDa BoNT/A complex, the 500-kDa BoNT/A complex, the 300-kDa BoNT/A complex. Non-limiting examples of the about 150 kDa BoNT/A neurotoxin include, e.g., SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4.

[0091] As used herein, the term "naturally occurring BoNT/A" refers to any BoNT/A produced by a naturally-occurring process, including, without limitation, BoNT/A isoforms produced from a post-translational modification, an alternatively-spliced transcript, or a spontaneous mutation, and BoNT/A subtypes, such as, e.g., a BoNT/A1 subtype, BoNT/A2 subtype, BoNT/A3 subtype, BoNT/A4 subtype, and BoNT/A5 subtype. A naturally occurring BoNT/A includes, without limitation, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or one that substitutes, deletes or adds, e.g., 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 amino acids from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. Commercially available pharmaceutical compositions of a naturally-occurring BoNT/A includes, without limitation, BOTOX.RTM. (Allergan, Inc., Irvine, Calif.), DYSPORT.RTM./RELOXIN.RTM., (Ipsen Ltd., Slough, England), PURTOX.RTM. (Mentor Corp., Santa Barbara, Calif.), XEOMIN.RTM. (Merz Pharmaceuticals, GmbH., Frankfurt, Germany), NEURONOX.RTM. (Medy-Tox, Inc., Ochang-myeon, South Korea), BTX-A.

[0092] As used herein, the term "non-naturally occurring BoNT/A" refers to any BoNT/A whose structure was modified with the aid of human manipulation, including, without limitation, a BoNT/A with an altered amino acid sequence produced by genetic engineering using random mutagenesis or rational design and a BoNT/A produced by in vitro chemical synthesis. Non-limiting examples of non-naturally occurring BoNT/As are described in, e.g., Steward, L. E. et al., Post-translational Modifications and Clostridial Neurotoxins, U.S. Pat. No. 7,223,577; Dolly, J. O. et al., Activatable Clostridial Toxins, U.S. Pat. No. 7,419,676; Steward, L. E. et al., Clostridial Neurotoxin Compositions and Modified Clostridial Neurotoxins, US 2004/0220386; Steward, L. E. et al., Modified Clostridial Toxins With Enhanced Targeting Capabilities For Endogenous Clostridial Toxin Receptor Systems, U.S. Patent Publication No. 2008/0096248; Steward, L. E. et al., Modified Clostridial Toxins With Altered Targeting Capabilities For Clostridial Toxin Target Cells, U.S. Patent Publication No. 2008/0161543; Steward, L. E. et al., Modified Clostridial Toxins With Enhanced Translocation Capabilities and Altered Targeting Activity For Clostridial Toxin Target Cells, U.S. Patent Publication No. 2008/0241881, each of which is hereby incorporated by reference in its entirety.

[0093] Thus in an embodiment, the BoNT/A activity being detected is from a naturally occurring BoNT/A. In aspects of this embodiment, the BoNT/A activity being detected is from a BoNT/A isoform or a BoNT/A subtype. In aspects of this embodiment, the BoNT/A activity being detected is from the BoNT/A of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. In other aspects of this embodiment, the BoNT/A activity being detected is from a BoNT/A having, e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% amino acid identity with SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. In other aspects of this embodiment, the BoNT/A activity being detected is from BOTOX.RTM., DYSPORT.RTM./RELOXIN.RTM., PURTOX.RTM., XEOMIN.RTM., NEURONOX.RTM., or BTX-A.

[0094] In another embodiment, the BoNT/A activity being detected is from a non-naturally occurring BoNT/A. In other aspects of this embodiment, the BoNT/A activity being detected is from a non-naturally occurring BoNT/A variant having, e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% amino acid identity with SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. In other aspects of this embodiment, the BoNT/A activity being detected is from a non-naturally occurring BoNT/A variant having, e.g., 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or more non-contiguous amino acid substitutions, deletions, or additions relative to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. In yet other aspects of this embodiment, the BoNT/A activity being detected is from a non-naturally occurring BoNT/A variant having, e.g., 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or more contiguous amino acid substitutions, deletions, or additions relative to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.

[0095] Aspects of the present disclosure comprise, in part, a SNAP-25. As used herein, the term "SNAP-25" refers to a naturally-occurring SNAP-25 or a non-naturally occurring SNAP-25 which is preferentially cleaved by a BoNT/A. As used herein, the term "preferentially cleaved" refers to that the cleavage rate of BoNT/A substrate by a BoNT/A is at least one order of magnitude higher than the cleavage rate of any other substrate by BoNT/A. In aspects of this embodiment, the cleavage rate of BoNT/A substrate by a BoNT/A is at least two orders of magnitude higher, at least three orders of magnitude higher, at least four orders of magnitude higher, or at least five orders of magnitude higher then that the cleavage rate of any other substrate by BoNT/A.

[0096] As used herein, the term "naturally occurring SNAP-25" refers to any SNAP-25 produced by a naturally-occurring process, including, without limitation, SNAP-25 isoforms produced from a post-translational modification, an alternatively-spliced transcript, or a spontaneous mutation, and SNAP-25 subtypes. A naturally occurring SNAP-25 includes, without limitation, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24, or one that substitutes, deletes or adds, e.g., 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or more amino acids from SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24.

[0097] As used herein, the term "non-naturally occurring SNAP-25" refers to any SNAP-25 whose structure was modified with the aid of human manipulation, including, without limitation, a SNAP-25 produced by genetic engineering using random mutagenesis or rational design and a SNAP-25 produced by in vitro chemical synthesis. Non-limiting examples of non-naturally occurring SNAP-25s are described in, e.g., Steward, L. E. et al., FRET Protease Assays for Clostridial Toxins, U.S. Pat. No. 7,332,567; Fernandez-Salas et al., Lipohilic Dye-based FRET Assays for Clostridial Toxin Activity, U.S. Patent Publication 2008/0160561, each of which is hereby incorporated by reference in its entirety. A non-naturally occurring SNAP-25 may substitute, delete or add, e.g., 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or more amino acids from SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24.

[0098] Thus in an embodiment, a SNAP-25 is a naturally occurring SNAP-25. In aspects of this embodiment, the SNAP-25 is a SNAP-25 isoform or a SNAP-25 subtype. In aspects of this embodiment, the naturally occurring SNAP-25 is the naturally occurring SNAP-25 of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24. In other aspects of this embodiment, the SNAP-25 is a naturally occurring SNAP-25 having, e.g., at least 70% amino acid identity, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% amino acid identity with SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24.

[0099] In another embodiment, a SNAP-25 is a non-naturally occurring SNAP-25. In other aspects of this embodiment, the SNAP-25 is a non-naturally occurring SNAP-25 having, e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% amino acid identity with SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. In other aspects of this embodiment, the SNAP-25 is a non-naturally occurring SNAP-25 having, e.g., 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or more non-contiguous amino acid substitutions, deletions, or additions relative to SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24. In yet other aspects of this embodiment, the SNAP-25 is a non-naturally occurring SNAP-25 having, e.g., 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or more contiguous amino acid substitutions, deletions, or additions relative to SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24.

[0100] A SNAP-25 can be an endogenous SNAP-25 or an exogenous SNAP-25. As used herein, the term "endogenous SNAP-25" refers to a SNAP-25 naturally present in the cell because it is naturally encoded within the cell's genome, such that the cell inherently expresses the SNAP-25 without the need an external source of SNAP-25 or an external source of genetic material encoding a SNAP-25. The expression of an endogenous SNAP-25 may be with or without environmental stimulation such as, e.g., cell differentiation. By definition, an endogenous SNAP-25 can only be a naturally-occurring SNAP-25 or variants thereof. For example, the following established cell lines express an endogenous SNAP-25: BE(2)-M17, Kelly, LA1-55n, N1E-115, N4TG3, N18, Neuro-2a, NG108-15, PC12, SH-SYSY, SiMa, and SK-N-BE(2)-C.

[0101] As used herein, the term "exogenous SNAP-25" refers to a SNAP-25 expressed in a cell through the introduction of an external source of SNAP-25 or an external source of genetic material encoding a SNAP-25 by human manipulation. The expression of an exogenous SNAP-25 may be with or without environmental stimulation such as, e.g., cell differentiation. As a non-limiting example, cells from an established cell line can express an exogenous SNAP-25 by transient or stably transfection of a SNAP-25. As another non-limiting example, cells from an established cell line can express an exogenous SNAP-25 by protein transfection of a SNAP-25. An exogenous SNAP-25 can be a naturally-occurring SNAP-25 or variants thereof, or a non-naturally occurring SNAP-25 or variants thereof.

[0102] Thus in an embodiment, cells from an established cell line express an endogenous SNAP-25. In aspects of this embodiment, the endogenous SNAP-25 expressed by cells from an established cell line is a naturally-occurring SNAP-25. In other aspects of this embodiment, the endogenous SNAP-25 expressed by cells from an established cell line is SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24. In yet aspects of this embodiment, the endogenous SNAP-25 expressed by cells from an established cell line is a naturally occurring SNAP-25, such as, e.g., a SNAP-25 isoform or a SNAP-25 subtype. In other aspects of this embodiment, the endogenous SNAP-25 expressed by cells from an established cell line is a naturally occurring SNAP-25 having, e.g., at least 70% amino acid identity, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% amino acid identity with SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24.

[0103] In another embodiment, cells from an established cell line are transiently or stably engineered to express an exogenous SNAP-25. In an aspect of this embodiment, cells from an established cell line are transiently or stably engineered to express a naturally-occurring SNAP-25. In other aspects of this embodiment, cells from an established cell line are transiently or stably engineered to express the naturally-occurring SNAP-25 of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24. In yet other aspects of this embodiment, cells from an established cell line are transiently or stably engineered to express a naturally occurring SNAP-25, such as, e.g., a SNAP-25 isoform or a SNAP-25 subtype. In still other aspects of this embodiment, cells from an established cell line are transiently or stably engineered to express a naturally occurring SNAP-25 having, e.g., at least 70% amino acid identity, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% amino acid identity with SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24.

[0104] In another aspect of the embodiment, cells from an established cell line are transiently or stably engineered to express a non-naturally occurring SNAP-25. In other aspects of this embodiment, cells from an established cell line are transiently or stably engineered to express a non-naturally occurring SNAP-25 having, e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% amino acid identity with SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24. In other aspects of this embodiment, cells from an established cell line are transiently or stably engineered to express a non-naturally occurring SNAP-25 having, e.g., 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or more non-contiguous amino acid substitutions, deletions, or additions relative to SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24. In yet other aspects of this embodiment, cells from an established cell line are transiently or stably engineered to express a non-naturally occurring SNAP-25 having, e.g., 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or more contiguous amino acid substitutions, deletions, or additions relative to SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24.

[0105] Assays that detect the cleavage of a BoNT/A substrate after exposure to a BoNT/A can be used to assess whether a cell is expressing an endogenous or exogenous SNAP-25. In these assays, generation of a SNAP-25 cleavage-product would be detected in cells expressing a SNAP-25 after BoNT/A treatment. Non-limiting examples of specific Western blot analysis, as well as well-characterized reagents, conditions and protocols are readily available from commercial vendors that include, without limitation, Amersham Biosciences, Piscataway, N.J.; Bio-Rad Laboratories, Hercules, Calif.; Pierce Biotechnology, Inc., Rockford, Ill.; Promega Corporation, Madison, Wis., and Stratagene, Inc., La Jolla, Calif. It is understood that these and similar assays for SNAP-25 cleavage can be useful in identifying cells expressing an endogenous or an exogenous SNAP-25.

[0106] As non-limiting examples, Western blot analysis using an antibody that recognizes BoNT/A SNAP-25-cleaved product or both the cleaved and uncleaved forms of SNAP-25 can be used to assay for uptake of BoNT/A. Examples of .alpha.-SNAP-25 antibodies useful for these assays include, without limitation, .alpha.-SNAP-25 mouse monoclonal antibody SMI-81 (Sternberger Monoclonals Inc., Lutherville, Md.), mouse .alpha.-SNAP-25 monoclonal antibody CI 71.1 (Synaptic Systems, Goettingen, Germany), .alpha.-SNAP-25 mouse monoclonal antibody CI 71.2 (Synaptic Systems, Goettingen, Germany), .alpha.-SNAP-25 mouse monoclonal antibody SP12 (Abcam, Cambridge, Mass.), .alpha.-SNAP-25 rabbit polyclonal antiserum (Synaptic Systems, Goettingen, Germany), .alpha.-SNAP-25 rabbit polyclonal antiserum (Abcam, Cambridge, Mass.), and .alpha.-SNAP-25 rabbit polyclonal antiserum S9684 (Sigma, St Louis, Mo.).

[0107] Aspects of the present disclosure comprise, in part, a BoNT/A receptor. As used herein, the term "BoNT/A receptor" refers to either a naturally-occurring BoNT/A receptor or a non-naturally occurring BoNT/A receptor which preferentially interacts with BoNT/A in a manner that elicits a BoNT/A intoxication response. As used herein, the term "preferentially interacts" refers to that the equilibrium dissociation constant (KD) of BoNT/A for a BoNT/A receptor is at least one order of magnitude less than that of BoNT/A for any other receptor at the cell surface. The equilibrium dissociation constant, a specific type of equilibrium constant that measures the propensity of an BoNT/A-BoNT/A receptor complex to separate (dissociate) reversibly into its component molecules, namely the BoNT/A and the BoNT/A receptor, is defined as KD=Ka/Kd at equilibrium. The association constant (Ka) is defined as Ka=[C]/[L][R] and the disassociation constant (Kd) is defined as Kd=[L][R]/[C], where [L] equals the molar concentration of BoNT/A, [R] is the molar concentration of a BoNT/A receptor, and [C] is the molar concentration of the BoNT/A-BoNT/A receptor complex, and where all concentrations are of such components when the system is at equilibrium. The smaller the dissociation constant, the more tightly bound the BoNT/A is to its receptor, or the higher the binding affinity between BoNT/A and BoNT/A receptor. In aspects of this embodiment, the disassociation constant of BoNT/A for a BoNT/A receptor is at least two orders of magnitude less, at least three orders of magnitude less, at least four orders of magnitude less, or at least five orders of magnitude less than that of BoNT/A for any other receptor. In other aspects of this embodiment, the binding affinity of a BoNT/A that preferentially interacts with a BoNT/A receptor can have an equilibrium disassociation constant (KD) of, e.g., of 500 nM or less, 400 nM or less, 300 nM or less, 200 nM, or less 100 nM or less. In other aspects of this embodiment, the binding affinity of a BoNT/A that preferentially interacts with a BoNT/A receptor can have an equilibrium disassociation constant (KD) of, e.g., of 90 nM or less, 80 nM or less, 70 nM or less, 60 nM, 50 nM or less, 40 nM or less, 30 nM or less, 20 nM, or less 10 nM or less. As used herein, the term "elicits a BoNT/A intoxication response" refers to the ability of a BoNT/A receptor to interact with a BoNT/A to form a neurotoxin/receptor complex and the subsequent internalization of that complex into the cell cytoplasm.

[0108] As used herein, the term "naturally occurring BoNT/A receptor" refers to any BoNT/A receptor produced by a naturally-occurring process, including, without limitation, BoNT/A receptor isoforms produced from a post-translational modification, an alternatively-spliced transcript, or a spontaneous mutation, and BoNT/A receptor subtypes. A naturally occurring BoNT/A receptor includes, without limitation, a fibroblast growth factor receptor 2 (FGFR2), a fibroblast growth factor receptor 3 (FGFR3), a synaptic vesicle glycoprotein 2 (SV2), and a complex ganglioside like GT1b, such as those described in Ester Fernandez-Salas, et al., Botulinum Toxin Screening Assays, U.S. Patent Publication 2008/0003240; Ester Fernandez-Salas, et al., Botulinum Toxin Screening Assays, U.S. Patent Publication 2008/0182799; Min Dong et al., SV2 is the Protein Receptor for Botulinum Neurotoxin A, Science (2006); S. Mahrhold et al, The Synaptic Vesicle Protein 2C Mediates the Uptake of Botulinum Neurotoxin A into Phrenic Nerves, 580(8) FEBS Lett. 2011-2014 (2006), each of which is hereby incorporated by reference in its entirety. A naturally occurring FGFR2 includes, without limitation, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, and SEQ ID NO: 70, or one that substitutes, deletes or adds, e.g., 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or more amino acids from SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, and SEQ ID NO: 70. A naturally occurring FGFR3 includes, without limitation, SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27, or one that substitutes, deletes or adds, e.g., 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or more amino acids from SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27. A naturally occurring SV2 includes, without limitation, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 31, or one that substitutes, deletes or adds, e.g., 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or more amino acids from SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 31.

[0109] As used herein, the term "non-naturally occurring BoNT/A receptor variant" refers to any BoNT/A receptor produced with the aid of human manipulation or design, including, without limitation, a BoNT/A receptor produced by genetic engineering using random mutagenesis or rational design and a BoNT/A receptor produced by chemical synthesis. Non-limiting examples of non-naturally occurring BoNT/A variants include, e.g., conservative BoNT/A receptor variants, non-conservative BoNT/A receptor variants, BoNT/A receptor chimeric variants and active BoNT/A receptor fragments.

[0110] As used herein, the term "non-naturally occurring BoNT/A receptor" refers to any BoNT/A receptor whose structure was modified with the aid of human manipulation, including, without limitation, a BoNT/A receptor produced by genetic engineering using random mutagenesis or rational design and a BoNT/A receptor produced by in vitro chemical synthesis. Non-limiting examples of non-naturally occurring BoNT/A receptors are described in, e.g., Ester Fernandez-Salas, et al., Botulinum Toxin Screening Assays, U.S. Patent Publication 2008/0003240; Ester Fernandez-Salas, et al., Botulinum Toxin Screening Assays, U.S. Patent Publication 2008/0182799, each of which is hereby incorporated by reference in its entirety. A non-naturally occurring BoNT/A receptor may substitute, delete or add, e.g., 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or more amino acids from SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, or SEQ ID NO: 70.

[0111] Thus in an embodiment, a BoNT/A receptor is a naturally occurring BoNT/A receptor such as, e.g., FGFR2, FGFR3 or SV2. In aspects of this embodiment, the BoNT/A receptor is a BoNT/A receptor isoform or a BoNT/A receptor subtype. In aspects of this embodiment, the naturally occurring BoNT/A receptor is the naturally occurring BoNT/A receptor of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, or SEQ ID NO: 70. In other aspects of this embodiment, the BoNT/A receptor is a naturally occurring BoNT/A receptor having, e.g., at least 70% amino acid identity, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% amino acid identity with SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, or SEQ ID NO: 70.

[0112] In another embodiment, a BoNT/A receptor is a non-naturally occurring BoNT/A receptor, such as, e.g., a genetically-engineered FGFR2, a genetically-engineered FGFR3, or a genetically-engineered SV2. In other aspects of this embodiment, the BoNT/A receptor is a non-naturally occurring BoNT/A receptor having, e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% amino acid identity with SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, or SEQ ID NO: 70. In other aspects of this embodiment, the BoNT/A receptor is a non-naturally occurring BoNT/A receptor having, e.g., 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or more non-contiguous amino acid substitutions, deletions, or additions relative to SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, or SEQ ID NO: 70. In yet other aspects of this embodiment, the BoNT/A receptor is a non-naturally occurring BoNT/A receptor having, e.g., 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or more contiguous amino acid substitutions, deletions, or additions relative to SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, or SEQ ID NO: 70.

[0113] A BoNT/A receptor can be an endogenous BoNT/A receptor or an exogenous BoNT/A receptor. As used herein, the term "endogenous BoNT/A receptor" refers to a BoNT/A receptor naturally present in the cell because it is naturally encoded within the cell's genome, such that the cell inherently expresses the BoNT/A receptor without the need for an external source of BoNT/A receptor or an external source of genetic material encoding a BoNT/A receptor. Expression of an endogenous BoNT/A receptor may be with or without environmental stimulation such as e.g., cell differentiation or promoter activation. For example, the following established cell lines express at least one endogenous BoNT/A receptor: BE(2)-M17, Kelly, LA1-55n, N1E-115, N4TG3, N18, Neuro-2a, NG108-15, PC12, SH-SYSY, SiMa, and SK-N-BE(2)-C. An endogenous BoNT/A receptor can only be a naturally-occurring BoNT/A receptor or naturally-occurring variants thereof.

[0114] As used herein, the term "exogenous BoNT/A receptor" refers to a BoNT/A receptor expressed in a cell through the introduction of an external source of BoNT/A receptor or an external source of genetic material encoding a BoNT/A receptor by human manipulation. The expression of an exogenous BoNT/A receptor may be with or without environmental stimulation such as, e.g., cell differentiation or promoter activation. As a non-limiting example, cells from an established cell line can express one or more exogenous BoNT/A receptors by transient or stably transfection of a polynucleotide molecule encoding a BoNT/A receptor, such as, e.g., a FGFR2, a FGFR3, or a SV2. As another non-limiting example, cells from an established cell line can express one or more exogenous BoNT/A receptors by protein transfection of the BoNT/A receptors, such as, e.g., a FGFR2, a FGFR3, or a SV2. An exogenous BoNT/A receptor can be a naturally-occurring BoNT/A receptor or naturally occurring variants thereof, or non-naturally occurring BoNT/A receptor or non-naturally occurring variants thereof.

[0115] Thus in an embodiment, cells from an established cell line express an endogenous BoNT/A receptor. In aspects of this embodiment, the endogenous BoNT/A receptor expressed by cells from an established cell line is a naturally-occurring BoNT/A receptor. In other aspects of this embodiment, the endogenous BoNT/A receptor expressed by cells from an established cell line is SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, or SEQ ID NO: 70. In yet aspects of this embodiment, the endogenous BoNT/A receptor expressed by cells from an established cell line is a naturally occurring BoNT/A receptor, such as, e.g., a BoNT/A receptor isoform or a BoNT/A receptor subtype. In other aspects of this embodiment, the endogenous BoNT/A receptor expressed by cells from an established cell line is a naturally occurring BoNT/A receptor having, e.g., at least 70% amino acid identity, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% amino acid identity with SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, or SEQ ID NO: 70.

[0116] In another embodiment, cells from an established cell line are transiently or stably engineered to express an exogenous BoNT/A receptor. In an aspect of this embodiment, cells from an established cell line are transiently or stably engineered to express a naturally-occurring BoNT/A receptor. In other aspects of this embodiment, cells from an established cell line are transiently or stably engineered to express the naturally-occurring BoNT/A receptor of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, or SEQ ID NO: 70. In yet other aspects of this embodiment, cells from an established cell line are transiently or stably engineered to express a naturally occurring BoNT/A receptor, such as, e.g., a BoNT/A receptor isoform or a BoNT/A receptor subtype. In still other aspects of this embodiment, cells from an established cell line are transiently or stably engineered to express a naturally occurring BoNT/A receptor having, e.g., at least 70% amino acid identity, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% amino acid identity with SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, or SEQ ID NO: 70.

[0117] In another aspect of the embodiment, cells from an established cell line are transiently or stably engineered to express a non-naturally occurring BoNT/A receptor. In other aspects of this embodiment, cells from an established cell line are transiently or stably engineered to express a non-naturally occurring BoNT/A receptor having, e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% amino acid identity with SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, or SEQ ID NO: 70. In other aspects of this embodiment, cells from an established cell line are transiently or stably engineered to express a non-naturally occurring BoNT/A receptor having, e.g., 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or more non-contiguous amino acid substitutions, deletions, or additions relative to SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, or SEQ ID NO: 70. In yet other aspects of this embodiment, cells from an established cell line are transiently or stably engineered to express a non-naturally occurring BoNT/A receptor having, e.g., 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or more contiguous amino acid substitutions, deletions, or additions relative to SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, or SEQ ID NO: 70

[0118] In another embodiment, cells from an established cell line are transiently or stably engineered to express an exogenous FGFR2, an exogenous FGFR3, an exogenous SV2, or any combination thereof. In aspects of this embodiment, cells from an established cell line are transiently or stably engineered to express a naturally-occurring FGFR2, a naturally-occurring FGFR3, a naturally-occurring SV2, or any combination thereof. In yet other aspects of this embodiment, cells from an established cell line are transiently or stably engineered to express a non-naturally-occurring FGFR2, a non-naturally-occurring FGFR3, a non-naturally-occurring SV2, or any combination thereof. In still other aspects of this embodiment, cells from an established cell line are transiently or stably engineered to express either a naturally-occurring FGFR2 or a non-naturally-occurring FGFR2, a naturally-occurring FGFR3 or a non-naturally-occurring FGFR3, a naturally-occurring SV2 or a non-naturally-occurring SV2, or any combination thereof.

[0119] Cells that express one or more endogenous or exogenous BoNT/A receptors can be identified by routine methods including direct and indirect assays for toxin uptake. Assays that determine BoNT/A binding or uptake properties can be used to assess whether a cell is expressing a BoNT/A receptor. Such assays include, without limitation, cross-linking assays using labeled BoNT/A, such as, e.g.,

[1251] BoNT/A,

[1251], see, e.g., Noriko Yokosawa et al., Binding of Clostridium botulinum type C neurotoxin to different neuroblastoma cell lines, 57(1) Infect. Immun. 272-277 (1989); Noriko Yokosawa et al., Binding of botulinum type Cl, D and E neurotoxins to neuronal cell lines and synaptosomes, 29(2) Toxicon 261-264 (1991); and Tei-ichi Nishiki et al., Identification of protein receptor for Clostridium botulinum type B neurotoxin in rat brain synaptosomes, 269(14) J. Biol. Chem. 10498-10503 (1994). Other non-limiting assays include immunocytochemical assays that detect BoNT/A binding using labeled or unlabeled antibodies, see, e.g., Atsushi Nishikawa et al., The receptor and transporter for internalization of Clostridium botulinum type C progenitor toxin into HT-29 cells, 319(2) Biochem. Biophys. Res. Commun. 327-333 (2004) and immunoprecipitation assays, see, e.g., Yukako Fujinaga et al., Molecular characterization of binding subcomponents of Clostridium botulinum type C progenitor toxin for intestinal epithelial cells and erythrocytes, 150(Pt 5) Microbiology 1529-1538 (2004), that detect bound toxin using labeled or unlabeled antibodies. Antibodies useful for these assays include, without limitation, antibodies selected against BoNT/A, antibodies selected against a BoNT/A receptor, such as, e.g., FGFR2, FGFR3, or SV2, and/or antibodies selected against a ganglioside, such as, e.g., GD1a, GD1b, GD3, GQ1b, or GT1b. If the antibody is labeled, the binding of the molecule can be detected by various means, including Western blot analysis, direct microscopic observation of the cellular location of the antibody, measurement of cell or substrate-bound antibody following a wash step, flow cytometry, electrophoresis or capillary electrophoresis, employing techniques well-known to those of skill in the art. If the antibody is unlabeled, one may employ a labeled secondary antibody for indirect detection of the bound molecule, and detection can proceed as for a labeled antibody. It is understood that these and similar assays that determine BoNT/A uptake properties or characteristics can be useful in identifying cells expressing endogenous or exogenous or BoNT/A receptors.

[0120] Assays that monitor the release of a molecule after exposure to BoNT/A can also be used to assess whether a cell is expressing one or more endogenous or exogenous BoNT/A receptors. In these assays, inhibition of the molecule's release would occur in cells expressing a BoNT/A receptor after BoNT/A treatment. Well known assays include methods that measure inhibition of radio-labeled catecholamine release from neurons, such as, e.g., [3H] noradrenaline or [3H] dopamine release, see e.g., A Fassio et al., Evidence for calcium-dependent vesicular transmitter release insensitive to tetanus toxin and botulinum toxin type F, 90(3) Neuroscience 893-902 (1999); and Sara Stigliani et al., The sensitivity of catecholamine release to botulinum toxin C1 and E suggests selective targeting of vesicles set into the readily releasable pool, 85(2) J. Neurochem. 409-421 (2003), or measures catecholamine release using a fluorometric procedure, see, e.g., Anton de Paiva et al., A role for the interchain disulfide or its participating thiols in the internalization of botulinum neurotoxin A revealed by a toxin derivative that binds to ecto-acceptors and inhibits transmitter release intracellularly, 268(28) J. Biol. Chem. 20838-20844 (1993); Gary W. Lawrence et al., Distinct exocytotic responses of intact and permeabilized chromaffin cells after cleavage of the 25-kDa synaptosomal-associated protein (SNAP-25) or synaptobrevin by botulinum toxin A or B, 236(3) Eur. J. Biochem. 877-886 (1996); and Patrick Foran et al., Botulinum neurotoxin C1 cleaves both syntaxin and SNAP-25 in intact and permeabilized chromaffin cells: correlation with its blockade of catecholamine release, 35(8) Biochemistry 2630-2636 (1996). Other non-limiting examples include assays that measure inhibition of hormone release from endocrine cells, such as, e.g., anterior pituitary cells or ovarian cells. It is understood that these and similar assays for molecule release can be useful in identifying cells expressing endogenous or exogenous or BoNT/A receptors.

[0121] Assays that detect the cleavage of a BoNT/A substrate after exposure to a BoNT/A can also be used to assess whether a cell is expressing one or more endogenous or exogenous BoNT/A receptors. In these assays, generation of a BoNT/A substrate cleavage-product, or disappearance of the intact BoNT/A substrate, would be detected in cells expressing a BoNT/A receptor after BoNT/A treatment. Non-limiting examples of specific Western blot analysis, as well as well-characterized reagents, conditions and protocols are readily available from commercial vendors that include, without limitation, Amersham Biosciences, Piscataway, N.J.; Bio-Rad Laboratories, Hercules, Calif.; Pierce Biotechnology, Inc., Rockford, Ill.; Promega Corporation, Madison, Wis., and Stratagene, Inc., La Jolla, Calif. It is understood that these and similar assays for BoNT/A substrate cleavage can be useful in identifying cells expressing endogenous or exogenous BoNT/A receptors.

[0122] As non-limiting examples, Western blot analysis using an antibody that recognizes BoNT/A SNAP-25-cleaved product or both the cleaved and uncleaved forms of SNAP-25 can be used to assay for uptake of BoNT/A. Examples of .alpha.-SNAP-25 antibodies useful for these assays include, without limitation, SMI-81 .alpha.-SNAP-25 mouse monoclonal antibody (Sternberger Monoclonals Inc., Lutherville, Md.), CI 71.1 mouse .alpha.-SNAP-25 monoclonal antibody (Synaptic Systems, Goettingen, Germany), CI 71.2 .alpha.-SNAP-25 mouse monoclonal antibody (Synaptic Systems, Goettingen, Germany), SP12 .alpha.-SNAP-25 mouse monoclonal antibody (Abcam, Cambridge, Mass.), .alpha.-SNAP-25 rabbit polyclonal antiserum (Synaptic Systems, Goettingen, Germany), .alpha.-SNAP-25 rabbit polyclonal antiserum S9684 (Sigma, St. Louis, Mo.), and .alpha.-SNAP-25 rabbit polyclonal antiserum (Abcam, Cambridge, Mass.).

[0123] Aspects of the present disclosure provide cells that through genetic manipulation or recombinant engineering are made to expresses an exogenous SNAP-25 and/or one or more exogenous BoNT/A receptors. Cells useful to express an exogenous SNAP-25 and/or one or more exogenous BoNT/A receptors through genetic manipulation or recombinant engineering include neuronal cells and non-neuronal cells that may or may not express an endogenous SNAP-25 and/or one or more endogenous BoNT/A receptors. It is further understood that such genetically manipulated or recombinantly engineered cells may express an exogenous SNAP-25 and one or more exogenous BoNT/A receptors under control of a constitutive, tissue-specific, cell-specific or inducible promoter element, enhancer element or both. It is understood that any cell is useful as long as the cell can be genetically manipulated or recombinantly engineered to expresses an exogenous SNAP-25 and/or one or more exogenous BoNT/A receptors and is capable of undergoing BoNT/A intoxication.

[0124] Methods useful for introducing into a cell an exogenous polynucleotide molecule encoding a component necessary for the cells to undergo the overall cellular mechanism whereby a BoNT/A proteolytically cleaves a SNAP-25 substrate, such as, e.g., a SNAP-25, a FGFR2, a FGFR3, or a SV2, include, without limitation, chemical-mediated delivery methods, such as, e.g., calcium phosphate-mediated, diethyl-aminoethyl (DEAE) dextran-mediated, lipid-mediated, polyethyleneimine (PEI)-mediated, polylysine-mediated and polybrene-mediated; physical-mediated delivery methods, such as, e.g., biolistic particle delivery, microinjection, protoplast fusion and electroporation; and viral-mediated delivery methods, such as, e.g., retroviral-mediated transfection, see e.g., Introducing Cloned Genes into Cultured Mammalian Cells, pp. 16.1-16.62 (Sambrook & Russell, eds., Molecular Cloning A Laboratory Manual, Vol. 3, 3.sup.rd ed. 2001); Alessia Colosimo et al., Transfer and Expression of Foreign Genes in Mammalian Cells, 29(2) Biotechniques 314-318, 320-322, 324 (2000); Philip Washbourne & A. Kimberley McAllister, Techniques for Gene Transfer into Neurons, 12(5) Curr. Opin. Neurobiol. 566-573 (2002); and Current Protocols in Molecular Biology, John Wley and Sons, pp 9.16.4-9.16.11 (2000), each of which is incorporated by reference in its entirety. One skilled in the art understands that selection of a specific method to introduce a polynucleotide molecule into a cell will depend, in part, on whether the cell will transiently or stably contain a component necessary for the cells to undergo the overall cellular mechanism whereby a BoNT/A proteolytically cleaves a SNAP-25 substrate. Non-limiting examples of polynucleotide molecule encoding a component necessary for the cells to undergo the overall cellular mechanism whereby a BoNT/A proteolytically cleaves a SNAP-25 substrate as as follows: FGFR2 polynucleotide molecule of SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, or SEQ ID NO: 138; FGFR3 polynucleotide molecule of SEQ ID NO: 139, SEQ ID NO: 140, or SEQ ID NO: 141; SV2 polynucleotide molecule of SEQ ID NO: 142, SEQ ID NO: 143, or SEQ ID NO: 144; and SNAP-25 polynucleotide molecule of SEQ ID NO: 145, or SEQ ID NO: 146.

[0125] Chemical-mediated delivery methods are well-known to a person of ordinary skill in the art and are described in, e.g., Martin Jordan & Florian Worm, Transfection of Adherent and Suspended Cells by Calcium Phosphate, 33(2) Methods 136-143 (2004); Chun Zhang et al., Polyethylenimine Strategies for Plasmid Delivery to Brain-Derived Cells, 33(2) Methods 144-150 (2004), each of which is hereby incorporated by reference in its entirety. Such chemical-mediated delivery methods can be prepared by standard procedures and are commercially available, see, e.g., CellPhect Transfection Kit (Amersham Biosciences, Piscataway, N.J.); Mammalian Transfection Kit, Calcium phosphate and DEAE Dextran, (Stratagene, Inc., La Jolla, Calif.); Lipofectamine.TM. Transfection Reagent (Invitrogen, Inc., Carlsbad, Calif.); ExGen 500 Transfection kit (Fermentas, Inc., Hanover, Md.), and SuperFect and Effectene Transfection Kits (Qiagen, Inc., Valencia, Calif.).

[0126] Physical-mediated delivery methods are well-known to a person of ordinary skill in the art and are described in, e.g., Jeike E. Biewenga et al., Plasmid-Mediated Gene Transfer in Neurons using the Biolistics Technique, 71(1) J. Neurosci. Methods. 67-75 (1997); John O'Brien & Sarah C. R. Lummis, Biolistic and Diolistic Transfection: Using the Gene Gun to Deliver DNA and Lipophilic Dyes into Mammalian Cells, 33(2) Methods 121-125 (2004); M. Golzio et al., In Vitro and In Vivo Electric Field-Mediated Permeabilization, Gene Transfer, and Expression, 33(2) Methods 126-135 (2004); and Oliver Gresch et al., New Non-Viral Method for Gene Transfer into Primary Cells, 33(2) Methods 151-163 (2004), each of which is hereby incorporated by reference in its entirety.

[0127] Viral-mediated delivery methods are well-known to a person of ordinary skill in the art and are described in, e.g., Chooi M. Lai et al., Adenovirus and Adeno-Associated Virus Vectors, 21(12) DNA Cell Biol. 895-913 (2002); Ilya Frolov et al., Alphavirus-Based Expression Vectors: Strategies and Applications, 93(21) Proc. Natl. Acad. Sci. U.S.A 11371-11377 (1996); Roland Wolkowicz et al., Lentiviral Vectors for the Delivery of DNA into Mammalian Cells, 246 Methods Mol. Biol. 391-411 (2004); A. Huser & C. Hofmann, Baculovirus Vectors: Novel Mammalian Cell Gene-Delivery Vehicles and Their Applications, 3(1) Am. J. Pharmacogenomics 53-63 (2003); Tiziana Tonini et al., Transient Production of Retroviral-and Lentiviral-Based Vectors for the Transduction of Mammalian Cells, 285 Methods Mol. Biol. 141-148 (2004); Manfred Gossen & Hermann Bujard, Tight Control of Gene Expression in Eukaryotic Cells by Tetracycline-Responsive Promoters, U.S. Pat. No. 5,464,758; Hermann Bujard & Manfred Gossen, Methods for Regulating Gene Expression, U.S. Pat. No. 5,814,618; David S. Hogness, Polynucleotides Encoding Insect Steroid Hormone Receptor Polypeptides and Cells Transformed With Same, U.S. Pat. No. 5,514,578; David S. Hogness, Polynucleotide Encoding Insect Ecdysone Receptor, U.S. Pat. No. 6,245,531; Elisabetta Vegeto et al., Progesterone Receptor Having C. Terminal Hormone Binding Domain Truncations, U.S. Pat. No. 5,364,791; Elisabetta Vegeto et al., Mutated Steroid Hormone Receptors, Methods for Their Use and Molecular Switch for Gene Therapy, U.S. Pat. No. 5,874,534, each of which is hereby incorporated by reference in its entirety. Such viral-mediated delivery methods can be prepared by standard procedures and are commercially available, see, e.g., ViraPower.TM. Adenoviral Expression System (Invitrogen, Inc., Carlsbad, Calif.) and ViraPower.TM. Adenoviral Expression System Instruction Manual 25-0543 version A, Invitrogen, Inc., (Jul. 15, 2002); and AdEasy.TM. Adenoviral Vector System (Stratagene, Inc., La Jolla, Calif.) and AdEasy.TM. Adenoviral Vector System Instruction Manual 064004f, Stratagene, Inc. Furthermore, such viral delivery systems can be prepared by standard methods and are commercially available, see, e.g., BD.TM. Tet-Off and Tet-On Gene Expression Systems (BD Biosciences-Clonetech, Palo Alto, Calif.) and BD.TM. Tet-Off and Tet-On Gene Expression Systems User Manual, PT3001-1, BD Biosciences Clonetech, (Mar. 14, 2003), GeneSwitch.TM. System (Invitrogen, Inc., Carlsbad, Calif.) and GeneSwitch.TM. System A Mifepristone-Regulated Expression System for Mammalian Cells version D, 25-0313, Invitrogen, Inc., (Nov. 4, 2002); ViraPower.TM. Lentiviral Expression System (Invitrogen, Inc., Carlsbad, Calif.) and ViraPower.TM. Lentiviral Expression System Instruction Manual 25-0501 version E, Invitrogen, Inc., (Dec. 8, 2003); and Complete Control.RTM. Retroviral Inducible Mammalian Expression System (Stratagene, La Jolla, Calif.) and Complete Control.RTM. Retroviral Inducible Mammalian Expression System Instruction Manual, 064005e.

[0128] Thus, in an embodiment, cells from an established cell line susceptible to BoNT/A intoxication transiently contain a polynucleotide molecule encoding a component necessary for the cells to undergo the overall cellular mechanism whereby a BoNT/A proteolytically cleaves a SNAP-25 substrate. In another embodiment, cells from an established cell line susceptible to BoNT/A intoxication transiently contain a polynucleotide molecule encoding a plurality of components necessary for the cells to undergo the overall cellular mechanism whereby a BoNT/A proteolytically cleaves a SNAP-25 substrate. In aspects of this embodiment, cells from an established cell line susceptible to BoNT/A intoxication transiently contain a polynucleotide molecule encoding FGFR2, FGFR3, SV2 or SNAP-25. In aspects of this embodiment, cells from an established cell line susceptible to BoNT/A intoxication transiently contain the polynucleotide molecule encoding FGFR2 of SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, or SEQ ID NO: 138. In other aspects of this embodiment, cells from an established cell line susceptible to BoNT/A intoxication transiently contain the polynucleotide molecule encoding FGFR3 of SEQ ID NO: 139, SEQ ID NO: 140, or SEQ ID NO: 141. In yet other aspects of this embodiment, cells from an established cell line susceptible to BoNT/A intoxication transiently contain the polynucleotide molecule encoding SV2 of SEQ ID NO: 142, SEQ ID NO: 143, or SEQ ID NO: 144. In yet other aspects of this embodiment, cells from an established cell line susceptible to BoNT/A intoxication transiently contain the polynucleotide molecule encoding SNAP-25 of SEQ ID NO: 145, or SEQ ID NO: 146.

[0129] In another embodiment, cells from an established cell line susceptible to BoNT/A intoxication stably contain a polynucleotide molecule encoding a component necessary for the cells to undergo the overall cellular mechanism whereby a BoNT/A proteolytically cleaves a SNAP-25 substrate. In another embodiment, cells from an established cell line susceptible to BoNT/A intoxication stably contain a polynucleotide molecule encoding a plurality of components necessary for the cells to undergo the overall cellular mechanism whereby a BoNT/A proteolytically cleaves a SNAP-25 substrate. In aspects of this embodiment, cells from an established cell line susceptible to BoNT/A intoxication stably contain a polynucleotide molecule encoding FGFR2, FGFR3, SV2 or SNAP-25. In aspects of this embodiment, cells from an established cell line susceptible to BoNT/A intoxication stably contain the polynucleotide molecule encoding FGFR2 of SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, or SEQ ID NO: 138. In other aspects of this embodiment, cells from an established cell line susceptible to BoNT/A intoxication stably contain the polynucleotide molecule encoding FGFR3 of SEQ ID NO: 139, SEQ ID NO: 140, or SEQ ID NO: 141. In yet other aspects of this embodiment, cells from an established cell line susceptible to BoNT/A intoxication stably contain the polynucleotide molecule encoding SV2 of SEQ ID NO: 142, SEQ ID NO: 143, or SEQ ID NO: 144. In yet other aspects of this embodiment, cells from an established cell line susceptible to BoNT/A intoxication stably contain the polynucleotide molecule encoding SNAP-25 of SEQ ID NO: 145, or SEQ ID NO: 146.

[0130] As mentioned above, an exogenous component necessary for the cells to undergo the overall cellular mechanism whereby a BoNT/A proteolytically cleaves a SNAP-25 substrate, such as, e.g., a SNAP-25, a FGFR2, a FGFR3, or a SV2 disclosed in the present specification can be introduced into a cell. Any and all methods useful for introducing such an exogenous component with a delivery agent into a cell population can be useful with the proviso that this method transiently introduces the exogenous component disclosed in the present specification in at least 50% of the cells within a given cell population. Thus, aspects of this embodiment can include a cell population in which, e.g., at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the given cell population transiently contains an exogenous component necessary for the cells to undergo the overall cellular mechanism whereby a BoNT/A proteolytically cleaves a SNAP-25 substrate, such as, e.g., a SNAP-25, a FGFR2, a FGFR3, or a SV2 disclosed in the present specification. As used herein, the term "delivery agent" refers to any molecule that enables or enhances internalization of a covalently-linked, non-covalently-linked or in any other manner associated with a polypeptide into a cell. Thus, the term "delivery agent" encompasses, without limitation, proteins, peptides, peptidomimetics, small molecules, polynucleotide molecules, liposomes, lipids, viruses, retroviruses and cells that, without limitation, transport a covalently or non-covalently linked molecule to the cell membrane, cell cytoplasm or nucleus. It further is understood that the term "delivery agent" encompasses molecules that are internalized by any mechanism, including delivery agents which function via receptor mediated endocytosis and those which are independent of receptor mediated endocytosis.

[0131] A delivery agent can also be an agent that enables or enhances cellular uptake of a covalently linked component, like FGFR2, FGFR3, SV2, or SNAP-25, such as, e.g., by chemical conjugation or by genetically produced fusion proteins. Methods that covalently link delivery agents and methods of using such agents are described in, e.g., Steven F. Dowdy, Protein Transduction System and Methods of Use Thereof, International Publication No WO 00/34308; Gerard Chassaing & Alain Prochiantz, Peptides which can be Used as Vectors for the Intracellular Addressing of Active Molecules, U.S. Pat. No. 6,080,724; Alan Frankel et al., Fusion Protein Comprising TAT-derived Transport Moiert, U.S. Pat. No. 5,674,980; Alan Frankel et al., TAT-derived Transport Polypeptide Conjugates, U.S. Pat. No. 5,747,641; Alan Frankel et al., TAT-derived Transport Polypeptides and Fusion Proteins, U.S. Pat. No. 5,804,604; Peter F. J. O'Hare et al., Use of Transport Proteins, U.S. Pat. No. 6,734,167; Yao-Zhong Lin & Jack J. Hawiger, Method for Importing Biologically Active Molecules into Cells, U.S. Pat. No. 5,807,746; Yao-Zhong Lin & Jack J. Hawiger, Method for Importing Biologically Active Molecules into Cells, U.S. Pat. No. 6,043,339; Yao-Zhong Lin et al., Sequence and Method for Genetic Engineering of Proteins with Cell Membrane Translocating Activity, U.S. Pat. No. 6,248,558; Yao-Zhong Lin et al., Sequence and Method for Genetic Engineering of Proteins with Cell Membrane Translocating Activity, U.S. Pat. No. 6,432,680; Jack J. Hawiger et al., Method for Importing Biologically Active Molecules into Cells, U.S. Pat. No. 6,495,518; Yao-Zhong Lin et al., Sequence and Method for Genetic Engineering of Proteins with Cell Membrane Translocating Activity, U.S. Pat. No. 6,780,843; Jonathan B. Rothbard & Paul A Wender, Method and Composition for Enhancing Transport Across Biological Membranes, U.S. Pat. No. 6,306,993; Jonathan B. Rothbard & Paul A Wender, Method and Composition for Enhancing Transport Across Biological Membranes, U.S. Pat. No. 6,495,663; and Pamela B. Davis et al., Fusion Proteins for Protein Delivery, U.S. Pat. No. 6,287,817, each of which is incorporated by reference in its entirety.

[0132] A delivery agent can also be an agent that enables or enhances cellular uptake of a non-covalently associated component, like FGFR2, FGFR3, SV2c, or SNAP-25. Methods that function in the absence of covalent linkage and methods of using such agents are described in, e.g., Gilles Divita et al, Peptide-Mediated Transfection Agents and Methods of Use, U.S. Pat. No. 6,841,535; Philip L Feigner and Olivier Zelphati, Intracellular Protein Delivery Compositions and Methods of Use, U.S. Patent Publication No. 2003/0008813; and Michael Karas, Intracellular Delivery of Small Molecules, Proteins and Nucleic Acids, U.S. Patent Publication 2004/0209797, each of which is incorporated by reference in its entirety. Such peptide delivery agents can be prepared and used by standard methods and are commercially available, see, e.g. the CHARIOT.TM. Reagent (Active Motif, Carlsbad, Calif.); BIO-PORTER.RTM. Reagent (Gene Therapy Systems, Inc., San Diego, Calif.), BIO TREK.TM. Protein Delivery Reagent (Stratagene, La Jolla, Calif.), and PRO-JECT.TM. Protein Transfection Reagent (Pierce Biotechnology Inc., Rockford, Ill.).

[0133] Aspects of the present disclosure comprise, in part, a sample comprising a BoNT/A. As used herein, the term "sample comprising a BoNT/A" refers to any biological matter that contains or potentially contains an active BoNT/A. A variety of samples can be assayed according to a method disclosed in the present specification including, without limitation, purified, partially purified, or unpurified BoNT/A; recombinant single chain or di-chain toxin with a naturally or non-naturally occurring sequence; recombinant BoNT/A with a modified protease specificity; recombinant BoNT/A with an altered cell specificity; bulk BoNT/A; a formulated BoNT/A product, including, e.g., BOTOX.RTM., DYSPORT.RTM./RELOXIN.RTM., XEOMIN.RTM., PURTOX.RTM., NEURONOX.RTM., BTX-A and; cells or crude, fractionated or partially purified cell lysates from, e.g., bacteria, yeast, insect, or mammalian sources; blood, plasma or serum; raw, partially cooked, cooked, or processed foods; beverages; animal feed; soil samples; water samples; pond sediments; lotions; cosmetics; and clinical formulations. It is understood that the term sample encompasses tissue samples, including, without limitation, mammalian tissue samples, livestock tissue samples such as sheep, cow and pig tissue samples; primate tissue samples; and human tissue samples. Such samples encompass, without limitation, intestinal samples such as infant intestinal samples, and tissue samples obtained from a wound. As non-limiting examples, a method of detecting picomolar amounts of BoNT/A activity can be useful for determining the presence or activity of a BoNT/A in a food or beverage sample; to assay a sample from a human or animal, for example, exposed to a BoNT/A or having one or more symptoms of botulism; to follow activity during production and purification of bulk BoNT/A; to assay a formulated BoNT/A product used in pharmaceutical or cosmetics applications; or to assay a subject's blood serum for the presence or absence of neutralizing .alpha.-BoNT/A antibodies.

[0134] Thus, in an embodiment, a sample comprising a BoNT/A is a sample comprising any amount of a BoNT/A. In aspects of this embodiment, a sample comprising a BoNT/A comprises about 100 ng or less, about 10 ng or less, about 1 ng or less, about 100 pg or less, about 10 pg or less, or about 1 pg or less of a BoNT/A. In other aspects of this embodiment, a sample comprising a BoNT/A comprises about 1 pM or less, about 100 nM or less, about 10 nM or less, about 1 nM or less, about 100 pM or less, about 10 pM or less, about 1 pM or less, about 100 fM or less, about 10 fM or less, or about 1 fM or less of a BoNT/A.

[0135] Aspects of the present disclosure comprise, in part, isolating from the treated cell a SNAP-25 component comprising a SNAP-25 having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond. As used herein, the term "SNAP-25 component comprising a SNAP-25 having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond" refers to a cellular component containing the SNAP-25 cleavage product. It is envisioned that any method suitable for enriching or isolating a SNAP-25 component can be useful, including, without limitation, cell lysing protocols, spin-column purification protocols, immunoprecipitation, affinity purification, and protein chromatography.

[0136] Aspects of the present disclosure comprise, in part, an .alpha.-SNAP-25 antibody linked to a solid phase support. As used herein, the term "solid-phase support" is synonymous with "solid phase" and refers to any matrix that can be used for immobilizing an .alpha.-SNAP-25 antibody disclosed in the present specification. Non-limiting examples of solid phase supports include, e.g., a tube; a plate; a column; pins or "dipsticks"; a magnetic particle, a bead or other spherical or fibrous chromatographic media, such as, e.g., agarose, sepharose, silica and plastic; and sheets or membranes, such as, e.g., nitrocellulose and polyvinylidene fluoride (PVDF). The solid phase support can be constructed using a wide variety of materials such as, e.g., glass, carbon, polystyrene, polyvinylchloride, polypropylene, polyethylene, dextran, nylon, diazocellulose, or starch. The solid phase support selected can have a physical property that renders it readily separable from soluble or unbound material and generally allows unbound materials, such as, e.g., excess reagents, reaction by-products, or solvents, to be separated or otherwise removed (by, e.g., washing, filtration, centrifugation, etc.) from solid phase support-bound assay component. Non-limiting examples of how to make and use a solid phase supports are described in, e.g., Molecular Cloning, A Laboratory Manual, supra, (2001); and Current Protocols in Molecular Biology, supra, (2004), each of which is hereby incorporated by reference in its entirety.

[0137] Aspects of the present disclosure comprise, in part, detecting the presence of an antibody-antigen complex comprising an .alpha.-SNAP-25 antibody that selectively binds to a SNAP-25 epitope having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond and a SNAP-25 cleavage product having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond. It is envisioned that any detection system can be used to practice aspects of this disclosed immuno-based method, with the provision that the signal to noise ratio can distinguish to a statistically significant degree the signal from the antibody-antigen complex from the background signal. Non-limiting examples of immuno-based detection systems include immunoblot analysis, like Western blotting and dot-blotting, immunoprecipitation analysis, enzyme-linked immunosorbent analysis (ELISA), and sandwich ELISA. The detection of the signal can be achieved using autoradiography with imaging or phosphorimaging (AU), chemiluminescense (CL), electrochemiluminescence (ECL), bioluminescence (BL), fluorescence, resonance energy transfer, plane polarization, colormetric, or flow cytometry (FC). Descriptions of immuno-based detection systems are disclosed in, e.g., Michael M. Rauhut, Chemiluminescence, In Kirk-Othmer Concise Encyclopedia of Chemical Technology (Ed. Grayson, 3rd ed, John Wley and Sons, 1985); A. W. Knight, A Review of Recent Trends in Analytical Applications of Electrogenerated Chemiluminescence, Trends Anal. Chem. 18(1): 47-62 (1999); K. A. Fahnrich, et al., Recent Applications of Electrogenerated Chemiluminescence sin Chemical Analysis, Talanta 54(4): 531-559 (2001); Commonly Used Techniques in Molecular Cloning, pp. A8.1-A8-55 (Sambrook & Russell, eds., Molecular Cloning A Laboratory Manual, Vol. 3, 3.sup.rd ed. 2001); Detection Systems, pp. A9.1-A9-49 (Sambrook & Russell, eds., Molecular Cloning A Laboratory Manual, Vol. 3, 3.sup.rd ed. 2001); Electrogenerated Chemiluminescence, (Ed. Allen J. Bard, Marcel Dekker, Inc., 2004), each of which is hereby incorporated by reference in its entirety.

[0138] A sandwich ELISA (or sandwich immunoassay) is a method based on two antibodies, which bind to different epitopes on the antigen. A capture antibody having a high binding specificity for the antigen of interest, is bound to a solid surface. The antigen is then added followed by addition of a second antibody referred to as the detection antibody. The detection antibody binds the antigen to a different epitope than the capture antibody. The antigen is therefore `sandwiched` between the two antibodies. The antibody binding affinity for the antigen is usually the main determinant of immunoassay sensitivity. As the antigen concentration increases the amount of detection antibody increases leading to a higher measured response. To quantify the extent of binding different reporter systems can be used, such as, e.g., an enzyme attached to the secondary antibody and a reporter substrate where the enzymatic reaction forms a readout as the detection signal. The signal generated is proportional to the amount of target antigen present in the sample. The reporter substrate used to measure the binding event determines the detection mode. A spectrophotometric plate reader is used for colorimetric detection. Chemiluminescent and electrochemiluminescence substrates have been developed which further amplify the signal and can be read on a luminescent reader. The reporter can also be a fluorescent readout where the enzyme step of the assay is replaced with a fluorophore and the readout is then measured using a fluorescent reader. Reagents and protocols necessary to perform an ECL sandwich ELISA are commercially available, including, without exception, MSD sandwich ELISA-ECL detection platform (Meso Scale Discovery, Gaithersburg, Md.).

[0139] Thus, in an embodiment, detecting the presence of an antibody-antigen complex comprising an .alpha.-SNAP-25 antibody that selectively binds to a SNAP-25 epitope having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond and a SNAP-25 cleavage product having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond can be performed using an immuno-blot analysis, an immunoprecipitation analysis, an ELISA, or a sandwich ELISA. In aspects of this embodiment, the detection is performed using a AU, CL, ECL, or BL immuno-blot analysis, a AU, CL, ECL, BL, or FC immunoprecipitation analysis, a AU, CL, ECL, BL, or FC ELISA, or a AU, CL, ECL, BL, or FC sandwich ELISA.

[0140] Aspects of the present disclosure can be practiced in a singleplex or multiplex fashion. An immuno-based method of detecting BoNT/A activity practiced in a single-plex fashion is one that only detects the presence of an antibody-antigen complex comprising an .alpha.-SNAP-25 antibody and a SNAP-25 cleavage product having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond. An immuno-based method of detecting BoNT/A activity practiced in a multiplex fashion is one that concurrently detects the presence of two or more antibody-antigen complexes; one of which is the antibody-antigen complex comprising an .alpha.-SNAP-25 antibody and a SNAP-25 cleavage product having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond; and the other(s) of which is antibody-antigen complex to a second, third, fourth, etc. different protein. A second protein can be used, e.g., as an internal control to minimize sample to sample variability by normalizing the amount of .alpha.-SNAP-25/SNAP-25 antibody-antigen complex detected to the amount of antibody-antigen complex detected for the second protein. As such, the second protein is usually one that is consistently expressed by the cell, such as a house-keeping protein. Non-limiting examples of a useful second protein, include, e.g., a Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH), Syntaxin, cytokines. Methods of performing an immuno-based assay in a multiplex fashion are described in, e.g., U. B. Nielsen and B. H. Geierstanger, Multiplexed Sandwich Assays in s, J. Immunol. Methods. 290(1-2): 107-120 2004); R. Barry and M, Soloviev, Quantitative Protein Profiling using Antibody Arrays, Proteomics, 4(12): 3717-3726 (2004); M. M. Ling et al., Multiplexing Molecular Diagnostics and Immunoassays using Emerging Microarray Technologies, Expert Rev Mol Diagn. 7(1): 87-98 (2007); S. X. Leng et al., ELISA and Multiplex Technologies for Cytokine Measurement in Inflammation and Aging Research, J Gerontol A Biol Sci Med Sci. 63(8): 879-884 (2008), each of which is hereby incorporated by reference in its entirety.

[0141] Thus, in one embodiment, an immuno-based method of detecting BoNT/A activity practiced in a single-plex fashion by only detecting the presence of an antibody-antigen complex comprising an .alpha.-SNAP-25 antibody and a SNAP-25 cleavage product having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond. In another embodiment, immuno-based method of detecting BoNT/A activity practiced in a multiplex fashion by concurrently detecting the presence of an antibody-antigen complex comprising an .alpha.-SNAP-25 antibody and a SNAP-25 cleavage product having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond and at least one other antibody-antigen complex to a protein other than SNAP-25, such as, e.g., GAPDH or Syntaxin.

[0142] Aspects of the present disclosure provide, in part, a method of determining BoNT/A immunoresistance. As used herein, the term "BoNT/A immunoresistance" means a mammal that does not fully respond to a BoNT/A therapy, or shows a reduced beneficial effect of a BoNT/A therapy because the immune response of that mammal, either directly or indirectly, reduces the efficacy of the therapy. A non-limiting example of reduced efficacy would be the presence in a mammal of at least one neutralizing .alpha.-BoNT/A antibody that binds to a BoNT/A toxin in a manner that reduces or prevents the specificity or activity of the toxin. As used herein, the term "BoNT/A therapy" means a treatment, remedy, cure, healing, rehabilitation or any other means of counteracting something undesirable in a mammal requiring neuromodulation using a BoNT/A toxin or administering to a mammal one or more controlled doses of a medication, preparation or mixture of a BoNT/A toxin that has medicinal, therapeutic, curative, cosmetic, remedial or any other beneficial effect. BoNT/A therapy encompasses, without limitation, the use of any naturally occurring or modified fragment thereof, in any formulation, combined with any carrier or active ingredient and administered by any route of administration. An exemplary, well-known BoNT/A therapy is a BOTOX.RTM. therapy.

[0143] Aspects of the present disclosure provide, in part, a test sample obtained from a mammal being tested for the presence or absence of .alpha.-BoNT/A neutralizing antibodies. As used herein, the term "test sample" refers to any biological matter that contains or potentially contains at least one .alpha.-BoNT/A antibody. An .alpha.-BoNT/A antibody can be a neutralizing anti-BoNT/A antibody or a non-neutralizing anti-BoNT/A antibody. As used herein, the term "neutralizing anti-BoNT/A antibodies" means any .alpha.-BoNT/A antibody that will, under physiological conditions, bind to a region of a BoNT/A toxin in such a manner as to reduce or prevent the toxin from exerting its effect in a BoNT/A therapy. As used herein, the term "non-neutralizing .alpha.-BoNT/A antibodies" means any .alpha.-BoNT/A antibody that will, under physiological conditions, bind to a region of a BoNT/A toxin, but not prevent the toxin from exerting its effect in a BoNT/A therapy. It is envisioned that any and all samples that can contain .alpha.-BoNT/A antibodies can be used in this method, including, without limitation, blood, plasma, serum and lymph fluid. In addition, any and all organisms capable of raising .alpha.-BoNT/A antibodies against a BoNT/A toxin can serve as a source for a sample including, but not limited to, birds and mammals, including mice, rats, goats, sheep, horses, donkeys, cows, primates and humans. Non-limiting examples of specific protocols for blood collection and serum preparation are described in, e.g., Marjorie Schaub Di Lorenzo & Susan King Strasinger, BLOOD COLLECTION IN HEALTHCARE (F. A. Davis Company, 2001); and Diana Garza & Kathleen Becan-McBride, PHLEBOTOMY HANDBOOK: BLOOD COLLECTION ESSENTIALS (Prentice Hall, 6.sup.th ed., 2002). These protocols are routine procedures well within the scope of one skilled in the art and from the teaching herein. A test sample can be obtained from an organism prior to exposure to a BoNT/A toxin, after a single BoNT/A treatment, after multiple BoNT/A toxin treatments, before onset of resistance to a BoNT/A therapy, or after onset of resistance to a BoNT/A therapy.

[0144] Aspects of the present disclosure provide, in part, a control sample. As used herein, the term "control sample" means any sample in which the presence or absence of the test sample is known and includes both negative and positive control samples. Wth respect to neutralizing .alpha.-BoNT/A antibodies, a negative control sample can be obtained from an individual who had never been exposed to BoNT/A and may include, without limitation, a sample from the same individual supplying the test sample, but taken before undergoing a BoNT/A therapy; a sample taken from a different individual never been exposed to BoNT/A; a pooled sample taken from a plurality of different individuals never been exposed to BoNT/A. With respect to neutralizing .alpha.-BoNT/A antibodies, a positive control sample can be obtained from an individual manifesting BoNT/A immunoresistance and includes, without limitation, individual testing positive in a patient-based testing assays; individual testing positive in an in vivo bioassay; and individual showing hyperimmunity, e.g., a BoNT/A vaccinated individual.

[0145] It is further foreseen that .alpha.-BoNT/A antibodies can be purified from a sample. Anti-BoNT/A antibodies can be purified from a sample, using a variety of procedures including, without limitation, Protein A/G chromatography and affinity chromatography. Non-limiting examples of specific protocols for purifying antibodies from a sample are described in, e.g., ANTIBODIES: A LABORATORY MANUAL (Edward Harlow & David Lane, eds., Cold Spring Harbor Laboratory Press, 2.sup.nd ed. 1998); USING ANTIBODIES: A LABORATORY MANUAL: PORTABLE PROTOCOL No. I (Edward Harlow & David Lane, Cold Spring Harbor Laboratory Press, 1998); and MOLECULAR CLONING, A LABORATORY MANUAL, supra, (2001), which are hereby incorporated by reference. In addition, non-limiting examples of antibody purification methods as well as well-characterized reagents, conditions and protocols are readily available from commercial vendors that include, without limitation, Pierce Biotechnology, Inc., Rockford, Ill.; and Zymed Laboratories, Inc., South San Francisco, Calif. These protocols are routine procedures well within the scope of one skilled in the art.

[0146] Thus, in an embodiment, a sample comprises blood. In aspect of this embodiment, the sample comprises mouse blood, rat blood, goat blood, sheep blood, horse blood, donkey blood, cow blood, primate blood or human blood. In another embodiment, a sample comprises plasma. In an aspect of this embodiment, a test sample comprises mouse plasma, rat plasma, goat plasma, sheep plasma, horse plasma, donkey plasma, cow plasma, primate plasma or human plasma. In another embodiment, a sample comprises serum. In an aspect of this embodiment, the sample comprises mouse serum, rat serum, goat serum, sheep serum, horse serum, donkey serum, cow serum, primate serum and human serum. In another embodiment, a sample comprises lymph fluid. In aspect of this embodiment, a sample comprises mouse lymph fluid, rat lymph fluid, goat lymph fluid, sheep lymph fluid, horse lymph fluid, donkey lymph fluid, cow lymph fluid, primate lymph fluid or human lymph fluid. In yet another embodiment, a sample is a test sample. In yet another embodiment, a sample is a control sample. In aspects of this embodiment, a control sample is a negative control sample or a positive control sample.

[0147] Aspects of the present disclosure provide, in part, comparing the amount of SNAP-25 having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond detected in step (d) to the amount of SNAP-25 having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond detected in step (e). In an embodiment, the amount of SNAP-25 cleavage product in the test sample is higher as compared to the amount of SNAP-25 cleavage product in the control sample. In an aspect of this embodiment, a higher amount of SNAP-25 cleavage product in the test sample as compared to a positive control sample indicates a reduction in or lack of BoNT/A immunoresistance in the mammal. In another aspect of this embodiment, an equivalent amount of SNAP-25 cleavage product in the test sample as compared to a negative control sample indicates a reduction in or lack of BoNT/A immunoresistance in the mammal. In another embodiment, the amount of SNAP-25 cleavage product in the test sample is lower as compared to the amount of SNAP-25 cleavage product in the control sample. In an aspect of this embodiment, a lower or equivalent amount of SNAP-25 cleavage product in the test sample as compared to a positive control sample indicates an increase in or presence of BoNT/A immunoresistance in the mammal. In another aspect of this embodiment, a lower amount of SNAP-25 cleavage product in the test sample as compared to a negative control sample indicates an increase in or presence of BoNT/A immunoresistance in the mammal.

[0148] It is envisioned that any and all assay conditions suitable for detecting the present of a neutralizing .alpha.-BoNT/A antibody in a sample are useful in the methods disclosed in the present specification, such as, e.g., linear assay conditions and non-linear assay conditions. In an embodiment, the assay conditions are linear. In an aspect of this embodiment, the assay amount of a BoNT/A is in excess. In another aspect of this embodiment, the assay amount of a BoNT/A is rate-limiting. In another aspect of this embodiment, the assay amount of a test sample is rate-limiting.

[0149] Aspects of the present disclosure can also be described as follows:

[0150] 1. A composition comprising a carrier linked to a flexible linker linked to SNAP-25 antigen having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond.

[0151] 2. The composition of 1, wherein the the P.sub.1 residue of the BoNT/A cleavage site scissile bond is glutamine or lysine.

[0152] 3. The composition of 1, wherein the SNAP-25 antigen comprises SEQ ID NO: 147.

[0153] 4. The composition of 1, wherein the flexible linker and the SNAP-25 antigen amino acid sequence is SEQ ID NO: 38 or SEQ ID NO: 46.

[0154] 5. An isolated .alpha.-SNAP-25 antibody, wherein the isolated .alpha.-SNAP-25 antibody binds an eptiope comprising a carboxyl-terminus at the P.sub.1 residue from the BoNT/A cleavage site scissile bond from a SNAP-25 cleavage product.

[0155] 6. The isolated .alpha.-SNAP-25 antibody of 5, wherein the .alpha.-SNAP-25 antibody has an association rate constant for an epitope not comprising a carboxyl-terminus glutamine of the BoNT/A cleavage site scissile bond from a SNAP-25 cleavage product of less than 1.times.10.sup.1 M.sup.-1 s.sup.-1; and wherein the .alpha.-SNAP-25 antibody has an equilibrium disassociation constant for the epitope of less than 0.450 nM.

[0156] 7. The isolated .alpha.-SNAP-25 antibody of 5, wherein the isolated .alpha.-SNAP-25 antibody has a heavy chain variable region comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 80, and SEQ ID NO: 82; and a light chain variable region comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, and SEQ ID NO: 92.

[0157] 8. The isolated .alpha.-SNAP-25 antibody of 5, wherein the isolated .alpha.-SNAP-25 antibody comprises at least the V.sub.H CDR1 of SEQ ID NO: 93, the V.sub.H CDR1 of SEQ ID NO: 94, the V.sub.H CDR1 of SEQ ID NO: 95, the V.sub.H CDR1 of SEQ ID NO: 118, the V.sub.H CDR1 of SEQ ID NO: 119, or the V.sub.H CDR1 of SEQ ID NO: 120.

[0158] 9. The isolated .alpha.-SNAP-25 antibody of 5, wherein the isolated .alpha.-SNAP-25 antibody comprises at least the V.sub.H CDR2 of SEQ ID NO: 96, the V.sub.H CDR2 of SEQ ID NO: 97, the V.sub.H CDR2 of SEQ ID NO: 98, the V.sub.H CDR2 of SEQ ID NO: 99, the V.sub.H CDR2 of SEQ ID NO: 121, the V.sub.H CDR2 of SEQ ID NO: 122, or the V.sub.H CDR2 of SEQ ID NO: 123.

[0159] 10. The isolated .alpha.-SNAP-25 antibody of 5, wherein the isolated .alpha.-SNAP-25 antibody comprises at least the V.sub.H CDR3 of SEQ ID NO: 100, the V.sub.H CDR3 of SEQ ID NO: 101, the V.sub.H CDR3 of SEQ ID NO: 102, or the V.sub.H CDR3 of SEQ ID NO: 124.

[0160] 11. The isolated .alpha.-SNAP-25 antibody of 5, wherein the isolated .alpha.-SNAP-25 antibody comprises at least the V.sub.L CDR1 of SEQ ID NO: 103, the V.sub.L CDR1 of SEQ ID NO: 104, the V.sub.L CDR1 of SEQ ID NO: 105, the V.sub.L CDR1 of SEQ ID NO: 106, the V.sub.L CDR1 of SEQ ID NO: 107, the V.sub.L CDR1 of SEQ ID NO: 125, the V.sub.L CDR1 of SEQ ID NO: 126, the V.sub.L CDR1 of SEQ ID NO: 127, the V.sub.L CDR1 of SEQ ID NO: 128, or the V.sub.L CDR1 of SEQ ID NO: 129.

[0161] 12. The isolated .alpha.-SNAP-25 antibody of 5, wherein the isolated .alpha.-SNAP-25 antibody comprises at least the V.sub.L CDR2 of SEQ ID NO: 108, the V.sub.L CDR2 of SEQ ID NO: 109, the V.sub.L CDR2 of SEQ ID NO: 110, the V.sub.L CDR2 of SEQ ID NO: 111, or the V.sub.L CDR2 of SEQ ID NO: 112.

[0162] 13. The isolated .alpha.-SNAP-25 antibody of 5, wherein the isolated .alpha.-SNAP-25 antibody comprises at least the V.sub.L CDR3 of SEQ ID NO: 113, the V.sub.L CDR3 of SEQ ID NO: 114, the V.sub.L CDR3 of SEQ ID NO: 115, the V.sub.L CDR3 of SEQ ID NO: 116, or the V.sub.L CDR3 of SEQ ID NO: 117.

[0163] 14. The isolated .alpha.-SNAP-25 antibody of 5, wherein the isolated .alpha.-SNAP-25 antibody comprises a heavy chain variable region comprising SEQ ID NO: 93, SEQ ID NO: 121 and SEQ ID NO: 100; and a light chain variable region comprising SEQ ID NO: 105, SEQ ID NO: 110 and SEQ ID NO: 115.

[0164] 15. The isolated .alpha.-SNAP-25 antibody of 5, wherein the isolated .alpha.-SNAP-25 antibody selectively binds the SNAP-25 epitope of SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 147 or SEQ ID NO: 148.

[0165] 16. The isolated .alpha.-SNAP-25 antibody of 5, wherein the isolated .alpha.-SNAP-25 antibody selectively binds the SNAP-25 epitope of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, or SEQ ID NO: 44.

[0166] 17. A method of detecting BoNT/A activity, the method comprising the steps of: a) treating a cell from an established cell line with a sample comprising a BoNT/A, wherein the cell from an established cell line is susceptible to BoNT/A intoxication by a BoNT/A; b) isolating from the treated cell a SNAP-25 component comprising a SNAP-25 cleavage product having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond; c) contacting the SNAP-25 component with an .alpha.-SNAP-25 antibody, wherein the .alpha.-SNAP-25 antibody binds an eptiope comprising a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond from a SNAP-25 cleavage product; and d) detecting the presence of an antibody-antigen complex comprising the .alpha.-SNAP-25 antibody and the SNAP-25 cleavage product; wherein detection by the antibody-antigen complex is indicative of BoNT/A activity.

[0167] 18. A method of detecting BoNT/A activity, the method comprising the steps of: a) treating a cell from an established cell line with a sample comprising a BoNT/A, wherein the cell from an established cell line is susceptible to BoNT/A intoxication by a BoNT/A; b) isolating from the treated cell a SNAP-25 component comprising a SNAP-25 cleavage product having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond; c) contacting the SNAP-25 component with an .alpha.-SNAP-25 antibody linked to a solid phase support, wherein the .alpha.-SNAP-25 antibody binds an eptiope comprising a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond from a SNAP-25 cleavage product; and d) detecting the presence of an antibody-antigen complex comprising the .alpha.-SNAP-25 antibody and the SNAP-25 cleavage product; wherein detection by the antibody-antigen complex is indicative of BoNT/A activity.

[0168] 19. A method of detecting BoNT/A activity, the method comprising the steps of: a) treating a cell from an established cell line with a sample comprising a BoNT/A, wherein the cell from an established cell line is susceptible to BoNT/A intoxication by a BoNT/A; b) isolating from the treated cell a SNAP-25 component comprising a SNAP-25 cleavage product having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond; c) fixing the SNAP-25 component to a solid phase support; d) contacting the SNAP-25 component with an .alpha.-SNAP-25 antibody, wherein the .alpha.-SNAP-25 antibody binds an eptiope comprising a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond from a SNAP-25 cleavage product; and e) detecting the presence of an antibody-antigen complex comprising the .alpha.-SNAP-25 antibody and the SNAP-25 cleavage product; wherein detection by the antibody-antigen complex is indicative of BoNT/A activity.

[0169] 20. A method of detecting BoNT/A activity, the method comprising the steps of: a) treating a cell from an established cell line with a sample comprising a BoNT/A, wherein the cell from an established cell line can uptake BoNT/A; b) isolating from the treated cell a SNAP-25 component comprising a SNAP-25 cleavage product having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond; c) contacting the SNAP-25 component with an .alpha.-SNAP-25 antibody, wherein the .alpha.-SNAP-25 antibody binds an eptiope comprising a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond from a SNAP-25 cleavage product; and d) detecting the presence of an antibody-antigen complex comprising the .alpha.-SNAP-25 antibody and the SNAP-25 cleavage product; wherein detection by the antibody-antigen complex is indicative of BoNT/A activity.

[0170] 21. A method of detecting BoNT/A activity, the method comprising the steps of: a) treating a cell from an established cell line with a sample comprising a BoNT/A, wherein the cell from an established cell line can uptake BoNT/A; b) isolating from the treated cell a SNAP-25 component comprising a SNAP-25 cleavage product having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond; c) contacting the SNAP-25 component with an .alpha.-SNAP-25 antibody linked to a solid phase support, wherein the .alpha.-SNAP-25 antibody binds an eptiope comprising a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond from a SNAP-25 cleavage product; and d) detecting the presence of an antibody-antigen complex comprising the .alpha.-SNAP-25 antibody and the SNAP-25 cleavage product; wherein detection by the antibody-antigen complex is indicative of BoNT/A activity.

[0171] 22. A method of detecting BoNT/A activity, the method comprising the steps of: a) treating a cell from an established cell line with a sample comprising a BoNT/A, wherein the cell from an established cell line can uptake BoNT/A; b) isolating from the treated cell a SNAP-25 component comprising a SNAP-25 cleavage product having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond; c) fixing the SNAP-25 component to a solid phase support; d) contacting the SNAP-25 component with an .alpha.-SNAP-25 antibody, wherein the .alpha.-SNAP-25 antibody binds an eptiope comprising a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond from a SNAP-25 cleavage product; and e) detecting the presence of an antibody-antigen complex comprising the .alpha.-SNAP-25 antibody and the SNAP-25 cleavage product; wherein detection by the antibody-antigen complex is indicative of BoNT/A activity.

[0172] 23. A method of determining BoNT/A immunoresistance in a mammal comprising the steps of: a) adding a BoNT/A to a test sample obtained from a mammal being tested for the presence or absence of .alpha.-BoNT/A neutralizing antibodies; b) treating a cell from an established cell line with the test sample, wherein the cell from an established cell line is susceptible to BoNT/A intoxication; c) isolating from the treated cells a SNAP-25 component comprising a SNAP-25 cleavage product having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond; d) contacting the SNAP-25 component with an .alpha.-SNAP-25 antibody, wherein the .alpha.-SNAP-25 antibody binds an eptiope comprising a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond from a SNAP-25 cleavage product; e) detecting the presence of an antibody-antigen complex comprising the .alpha.-SNAP-25 antibody and the SNAP-25 cleavage product; f) repeating steps b-e with a negative control sample instead of a test sample, the negative control sample comprising a BoNT/A and a serum known not to contain .alpha.-BoNT/A neutralizing antibodies; and g) comparing the amount of antibody-antigen complex detected in step e to the amount of antibody-antigen complex detected in step f, wherein detection of a lower amount of antibody-antigen complex detected in step e relative to the amount of antibody-antigen complex detected in step f is indicative of the presence of .alpha.-BoNT/A neutralizing antibodies.

[0173] 24. A method of determining BoNT/A immunoresistance in a mammal comprising the steps of: a) adding a BoNT/A to a test sample obtained from a mammal being tested for the presence or absence of .alpha.-BoNT/A neutralizing antibodies; b) treating a cell from an established cell line with the test sample, wherein the cell from an established cell line is susceptible to BoNT/A intoxication; c) isolating from the treated cells a SNAP-25 component comprising a SNAP-25 cleavage product having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond; d) contacting the SNAP-25 component with an .alpha.-SNAP-25 antibody linked to a solid phase support, wherein the .alpha.-SNAP-25 antibody binds an eptiope comprising a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond from a SNAP-25 cleavage product; e) detecting the presence of an antibody-antigen complex comprising the .alpha.-SNAP-25 antibody and the SNAP-25 cleavage product; f) repeating steps b-e with a negative control sample instead of a test sample, the negative control sample comprising a BoNT/A and a serum known not to contain .alpha.-BoNT/A neutralizing antibodies; and g) comparing the amount of antibody-antigen complex detected in step e to the amount of antibody-antigen complex detected in step f, wherein detection of a lower amount of antibody-antigen complex detected in step e relative to the amount of antibody-antigen complex detected in step f is indicative of the presence of .alpha.-BoNT/A neutralizing antibodies.

[0174] 25. A method of determining BoNT/A immunoresistance in a mammal comprising the steps of: a) adding a BoNT/A to a test sample obtained from a mammal being tested for the presence or absence of .alpha.-BoNT/A neutralizing antibodies; b) treating a cell from an established cell line with the test sample, wherein the cell from an established cell line is susceptible to BoNT/A intoxication; c) isolating from the treated cells a SNAP-25 component comprising a SNAP-25 cleavage product having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond; d) fixing the SNAP-25 component to a solid phase support; e) contacting the SNAP-25 component with an .alpha.-SNAP-25 antibody, wherein the .alpha.-SNAP-25 antibody binds an eptiope comprising a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond from a SNAP-25 cleavage product; f) detecting the presence of an antibody-antigen complex comprising the .alpha.-SNAP-25 antibody and the SNAP-25 cleavage product; g) repeating steps b-f with a negative control sample instead of a test sample, the negative control sample comprising a BoNT/A and a serum known not to contain .alpha.-BoNT/A neutralizing antibodies; and h) comparing the amount of antibody-antigen complex detected in step f to the amount of antibody-antigen complex detected in step g, wherein detection of a lower amount of antibody-antigen complex detected in step f relative to the amount of antibody-antigen complex detected in step g is indicative of the presence of .alpha.-BoNT/A neutralizing antibodies.

[0175] 26. A method of determining BoNT/A immunoresistance in a mammal comprising the steps of: a) adding a BoNT/A to a test sample obtained from a mammal being tested for the presence or absence of .alpha.-BoNT/A neutralizing antibodies; b) treating a cell from an established cell line with the test sample, wherein the cell from an established cell line can uptake BoNT/A; c) isolating from the treated cells a SNAP-25 component comprising a SNAP-25 cleavage product having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond; d) contacting the SNAP-25 component with an .alpha.-SNAP-25 antibody, wherein the .alpha.-SNAP-25 antibody binds an eptiope comprising a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond from a SNAP-25 cleavage product; e) detecting the presence of an antibody-antigen complex comprising the .alpha.-SNAP-25 antibody and the SNAP-25 cleavage product; f) repeating steps b-e with a negative control sample instead of a test sample, the negative control sample comprising a BoNT/A and a serum known not to contain .alpha.-BoNT/A neutralizing antibodies; and g) comparing the amount of antibody-antigen complex detected in step e to the amount of antibody-antigen complex detected in step f, wherein detection of a lower amount of antibody-antigen complex detected in step e relative to the amount of antibody-antigen complex detected in step f is indicative of the presence of .alpha.-BoNT/A neutralizing antibodies.

[0176] 27. A method of determining BoNT/A immunoresistance in a mammal comprising the steps of: a) adding a BoNT/A to a test sample obtained from a mammal being tested for the presence or absence of .alpha.-BoNT/A neutralizing antibodies; b) treating a cell from an established cell line with the test sample, wherein the cell from an established cell line can uptake BoNT/A; c) isolating from the treated cells a SNAP-25 component comprising a SNAP-25 cleavage product having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond; d) contacting the SNAP-25 component with an .alpha.-SNAP-25 antibody linked to a solid phase support, wherein the .alpha.-SNAP-25 antibody binds an eptiope comprising a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond from a SNAP-25 cleavage product; e) detecting the presence of an antibody-antigen complex comprising the .alpha.-SNAP-25 antibody and the SNAP-25 cleavage product; f) repeating steps b-e with a negative control sample instead of a test sample, the negative control sample comprising a BoNT/A and a serum known not to contain .alpha.-BoNT/A neutralizing antibodies; and g) comparing the amount of antibody-antigen complex detected in step e to the amount of antibody-antigen complex detected in step f, wherein detection of a lower amount of antibody-antigen complex detected in step e relative to the amount of antibody-antigen complex detected in step f is indicative of the presence of .alpha.-BoNT/A neutralizing antibodies.

[0177] 28. A method of determining BoNT/A immunoresistance in a mammal comprising the steps of: a) adding a BoNT/A to a test sample obtained from a mammal being tested for the presence or absence of .alpha.-BoNT/A neutralizing antibodies; b) treating a cell from an established cell line with the test sample, wherein the cell from an established cell line can uptake BoNT/A; c) isolating from the treated cells a SNAP-25 component comprising a SNAP-25 cleavage product having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond; d) fixing the SNAP-25 component to a solid phase support; e) contacting the SNAP-25 component with an .alpha.-SNAP-25 antibody, wherein the .alpha.-SNAP-25 antibody binds an eptiope comprising a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond from a SNAP-25 cleavage product; f) detecting the presence of an antibody-antigen complex comprising the .alpha.-SNAP-25 antibody and the SNAP-25 cleavage product; g) repeating steps b-f with a negative control sample instead of a test sample, the negative control sample comprising a BoNT/A and a serum known not to contain .alpha.-BoNT/A neutralizing antibodies; and h) comparing the amount of antibody-antigen complex detected in step f to the amount of antibody-antigen complex detected in step g, wherein detection of a lower amount of antibody-antigen complex detected in step f relative to the amount of antibody-antigen complex detected in step g is indicative of the presence of .alpha.-BoNT/A neutralizing antibodies.

[0178] 29. The method of 17-22 and 23-25, wherein the cell is susceptible to BoNT/A intoxication by about 500 pM or less, by about 400 pM or less, by about 300 pM or less, by about 200 pM or less, by about 100 pM or less of a BoNT/A.

[0179] 30. The method of 20-22 and 26-28, wherein the cell can uptake about 500 pM or less, by about 400 pM or less, by about 300 pM or less, by about 200 pM or less, by about 100 pM or less of BoNT/A.

[0180] 31. The method of 17-22, wherein the sample comprises about 100 ng or less, about 10 ng or less, about 1 ng or less, 100 fg or less, 10 fg or less, or 1 fg or less of a BoNT/A

[0181] 32. The method of 17-22, wherein the sample comprises about 100 nM or less, about 10 nM or less, about 1 nM or less, about 100 pM or less, about 10 pM or less, about 1 pM or less, about 100 fM or less, about 10 fM or less, or about 1 fM or less of a BoNT/A.

[0182] 33. The method of 17-28, wherein the .alpha.-SNAP-25 antibody is the isolated .alpha.-SNAP-25 antibody of 5-16.

[0183] 34. The method of 17-28, wherein the presence of an antibody-antigen complex is detected by an immuno-blot analysis, an immunoprecipitation analysis, an ELISA, or a sandwich ELISA

[0184] 35. The method of 17-28, wherein the immuno-based method has a signal-to-noise ratio for the lower asymptote of at least 3:1, at least 5:1, at least 10:1, at least 20:1, at least 50:1, or at least 100:1.

[0185] 36. The method of 17-28, wherein the immuno-based method has a signal-to-noise ratio for the higher asymptote of at least 10:1, at least 20:1, at least 50:1, at least 100:1, at least 200:1, at least 300:1, at least 400:1, at least 500:1, or at least 600:1.

[0186] 37. The method of 17-28, wherein the immuno-based method can detect the EC.sub.50 activity of, e.g., at least 100 ng, at least 50 ng, at least 10 ng, at least 5 ng, at least 100 pg, at least 50 pg, at least 10 pg, at least 5 pg, at least 100 fg, at least 50 fg, at least 10 fg, or at least 5 fg.

[0187] 38. The method of 17-28, wherein the immuno-based method can detect the EC.sub.50 activity of, e.g., at least 10 nM, at least 5 nM, at least 100 pM, at least 50 pM, at least 10 pM, at least 5 pM, at least 100 fM, at least 50 fM, at least 10 fM, at least 5 fM, or at least 1 fM.

[0188] 39. The method of 17-28, wherein the immuno-based method has an LOD of, e.g., 10 pg or less, 9 pg or less, 8 pg or less, 7 pg or less, 6 pg or less, 5 pg or less, 4 pg or less, 3 pg or less, 2 pg or less, 1 pg or less of a BoNT/A

[0189] 40. The method of 17-28, wherein the immuno-based method has an LOD of, e.g., 100 fM or less, 90 fM or less, 80 fM or less, 70 fM or less, 60 fM or less, 50 fM or less, 40 fM or less, 30 fM or less, 20 fM or less, or 10 fM or less of a BoNT/A.

[0190] 41. The method of 17-28, wherein the immuno-based method has an LOQ of, e.g., 10 pg or less, 9 pg or less, 8 pg or less, 7 pg or less, 6 pg or less, 5 pg or less, 4 pg or less, 3 pg or less, 2 pg or less, 1 pg or less of a BoNT/A

[0191] 42. The method of 17-28, wherein the immuno-based method has an LOQ of, e.g., 100 fM or less, 90 fM or less, 80 fM or less, 70 fM or less, 60 fM or less, 50 fM or less, 40 fM or less, 30 fM or less, 20 fM or less, or 10 fM or less of a BoNT/A.

[0192] 43. The method of 17-28, wherein the immuno-based method can distinguish a fully-active BoNT/A from a partially-active BoNT/A having 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, or 10% or less the activity of a fully-active BoNT/A.

EXAMPLES

Example I

Screening of Candidate Cell Lines

[0193] The following example illustrates how to identify established cell lines susceptible to BoNT/A intoxication or have BoNT/A uptake capacity required for a method of detecting BoNT/A activity disclosed in the rpesent specification.

1. Growth of Stock Culture of Candidate Cell Lines.

[0194] To grow the cell lines, a suitable density of cells from the cell line being tested were plated in a 162 cm.sup.2 tissue culture flask containing 30 mL of a suitable growth medium (see Table 1), and grown in a 37.degree. C. incubator under 5% or 10% carbon dioxide until cells reached the desired density.

TABLE-US-00001 TABLE 1 Media Used in Cell Line Screening. Cell Line Serum Growth Media Composition Kelly RPMI 1640, 10% fetal bovine serum, 1% Penicillin- SiMa Streptomycin, 2 mM L-Glutamine NB69 RPMI 1640, 15% fetal bovine serum, 1% Penicillin- Streptomycin CHP-126 RPMI 1640, 20% fetal bovine serum, 1% Pencillin- Streptomycin N4TG3 RPMI 1640, 10% fetal bovine serum, 1% Pencillin- Streptomycin, 100 .mu.M 6-thioguanine MHH-NB-11 RPMI 1640, 10% fetal bovine serum, 1% Pencillin- Streptomycin, 2 mM L-glutamine, 0.1 mM non- essential amino acids PC12 RPMI 1640, 5% heat-inactivated fetal bovine serum, 10% equine serum, 2 mM GlutaMAX .TM., 10 mM HEPES, 1 mM sodium pyruvate, 1% Penicillin- Streptomycin N18TG2 DMEM (11885-084, Gibco), 10% fetal bovine serum, 1% Penicillin-Streptomycin, 100 .mu.M 6-thioguanine N1E-115 90% DMEM, 10% heat-inactivated fetal bovine serum, N18 2 mM Glutamine, 2 mM glucose ND8/34 NG108-15 NG115-401L NS20Y SK-N-SH SK-N-DZ 90% DMEM, 10% heat-inactivated fetal bovine serum, SK-N-F1 4 mM glucose, 0.1 mM non-essential amino acids, 1.5 g/L NaHCO.sub.3 BE(2)-C EMEM(11090-081, Gibco), Ham's F12 (11765-054, BE(2)-M17 Gibco), 10% heat-inactivated fetal bovine serum, 2 mM CHP-212 Glutamine, 0.1 mM non-essential amino acids, LA-1-55n LA-N-1 MC-1XC SK-N-BE(2) SH-SY5Y NB4 1A3 Ham's F10 (12471-017, Gibco), 2.5% heat-inactivated fetal bovine serum, 15% heat-inactivated horse serum, 2 mM Glutamine Neuro-2a EMEM, 10% heat-inactivated fetal bovine serum, 2 mM Glutamine, 0.1 mM non-essential amino acids, 1.5 g/L NaHCO.sub.3, 1 mM Sodium pyruvate

2. Single-Dose Screening of Candidate Cell Lines Using 1 nM BoNT/A.

[0195] One parameter tested to improve the sensitivity of a cell-based assay was to identify suitable cell lines that exhibited a good capacity to uptake a Clostridial neurotoxin and adhere to a substrate surface. Initially, cell lines were tested for their ability to uptake 1 nM BoNT/A and their ability to attach to a surface. To determine whether a cell line was able to uptake 1 nM BoNT/A, a suitable density of cells from a stock culture of the cell line being tested was plated into the wells of 24-well tissue culture plates containing 1 mL of an appropriate serum growth medium (Table 1). The cells were grown in a 37.degree. C. incubator under 5% carbon dioxide until cells reached the desired density (approximately 18 to 24 hours). The growth media was aspirated from each well and replaced with either 1) fresh growth media containing no toxin (untreated cell line) or 2) fresh growth media containing 1 nM of a BoNT/A complex (treated cell line). After an overnight incubation, the cells were washed by aspirating the growth media and rinsing each well with 200 .mu.l of 1.times.PBS. To harvest the cells, the 1.times.PBS was aspirated, the cells were lysed by adding 50 .mu.l of 2.times.SDS Loading Buffer, the lysate was transferred to a clean test tube and the sample was heated to 95.degree. C. for 5 minutes.

[0196] To detect for the presence of both uncleaved SNAP-25 substrate and cleaved SNAP-25 products, an aliquot from each harvested sample was analyzed by Western blot. In this analysis, a 12 .mu.l aliquot of the harvested sample was separated by MOPS polyacrylamide gel electrophoresis using NuPAGE.RTM. Novex 12% Bis-Tris precast polyacrylamide gels (Invitrogen Inc., Carlsbad, Calif.) under denaturing, reducing conditions. Separated peptides were transferred from the gel onto polyvinylidene fluoride (PVDF) membranes (Invitrogen Inc., Carlsbad, Calif.) by Western blotting using a Trans-Blot.RTM. SD semi-dry electrophoretic transfer cell apparatus (Bio-Rad Laboratories, Hercules, Calif.). PVDF membranes were blocked by incubating at room temperature for 2 hours in a solution containing Tris-Buffered Saline (TBS) (25 mM 2-amino-2-hydroxymethyl-1,3-propanediol hydrochloric acid (Tris-HCl)(pH 7.4), 137 mM sodium chloride, 2.7 mM potassium chloride), 0.1% TWEEN-20.RTM. (polyoxyethylene (20) sorbitan monolaureate), 2% Bovine Serum Albumin (BSA), 5% nonfat dry milk. Blocked membranes were incubated at 4.degree. C. for overnight in TBS, 0.1% TWEEN-20.RTM. (polyoxyethylene (20) sorbitan monolaureate), 2% BSA, and 5% nonfat dry milk containing either 1) a 1:5,000 dilution of an .alpha.-SNAP-25 mouse monoclonal antibody as the primary antibody (SMI-81; Sternberger Monoclonals Inc., Lutherville, Md.); or 2) a 1:5,000 dilution of S9684 .alpha.-SNAP-25 rabbit polyclonal antiserum as the primary antibody (Sigma, St. Louis, Mo.). Both .alpha.-SNAP-25 mouse monoclonal and rabbit polyclonal antibodies can detect both the uncleaved SNAP-25 substrate and the SNAP-25 cleavage product, allowing for the assessment of overall SNAP-25 expression in each cell line and the percent of SNAP-25 cleaved after BoNT/A treatment as a parameter to assess the amount of BoNT/A uptake. Primary antibody probed blots were washed three times for 15 minutes each time in TBS, TWEEN-20.RTM. (polyoxyethylene (20) sorbitan monolaureate). Washed membranes were incubated at room temperature for 2 hours in TBS, 0.1% TWEEN-20.RTM. (polyoxyethylene (20) sorbitan monolaureate), 2% BSA, and 5% nonfat dry milk containing either 1) a 1:10,000 dilution of goat polyclonal anti-mouse immunoglobulin G, heavy and light chains (IgG, H+L) antibody conjugated to horseradish peroxidase (Zymed, South San Francisco, Calif.) as a secondary antibody; or 2) a 1:10,000 dilution of goat polyclonal anti-rabbit immunoglobulin G, heavy and light chains (IgG, H+L) antibody conjugated to horseradish peroxidase (Zymed, South San Francisco, Calif.) as a secondary antibody. Secondary antibody-probed blots were washed three times for 15 minutes each time in TBS, 0.1% TWEEN-20.RTM. (polyoxyethylene (20) sorbitan monolaureate). Signal detection of the labeled SNAP-25 products were visualized using the ECL Plus.TM. Western Blot Detection System (GE Healthcare, Amersham Biosciences, Piscataway, N.J.) and the membrane was imaged and the percent of cleaved quantified with a Typhoon 9410 Variable Mode Imager and Imager Analysis software (GE Healthcare, Amersham Biosciences, Piscataway, N.J.). The choice of pixel size (100 to 200 pixels) and PMT voltage settings (350 to 600, normally 400) depended on the individual blot. Table 2 indicates the cell lines where a SNAP-25 cleavage product was detected when treated with 1 nM BoNT/A. The following cell lines exhibited both an uptake of 1 nM BoNT/A and appropriate attachment to a substrate surface: BE(2)-M17, IMR-32, Kelly, LA1-55n, N1E-115, N4TG3, N18, Neuro-2a, NG108-15, PC12, SH-SYSY, SiMa and SK-N-BE(2)-C.

[0197] To determine whether a cell line was able to attach to a surface, a suitable density of cells from a stock culture of the cell line being tested was plated into the wells of 24-well tissue culture plates containing 1 mL of an appropriate growth media (Table 1). The cells were grown in a 37.degree. C. incubator under 5% carbon dioxide until cells reach the desired density (approximately 18 to 24 hours). Cell attachment was assessed by the percentage of cells that adhered to the bottom well surface of the tissue plate relative to the total number of cells seeded. Cell lines CHP-126, IMR-32, LA-N-1, MC-IXC, NG115-401L, SK-N-BE(2)-C, SK-N-F1 and SK-N-MC were deemed unsuitable because each cell line exhibited less than 50% attachment (Table 2). All other cells lines tested exhibited suitable cell attachment characteristics (Table 2).

TABLE-US-00002 TABLE 2 Single-Dose Screening of Candidate Cell Lines Using 1 nM BoNT/A. 1 nM BoNT/A Cell Line Description Source Uptake Attachment BE(2)-C Human neuroblastoma ATCC CRL-2268 No >60% BE(2)-M17 Human neuroblastoma ATCC CRL-2267 Yes >60% CHP-126 Human neuroblastoma DSMZ ACC 304 No <50% CHP-212 Human neuroblastoma ATCC CRL-2273 No >60% HCN-1a Brain cortical neuron ATCC CRL-10442 No >60% HCN-2 Brain cortical neuron ATCC CRL-10742 No >60% IMR-32 Human neuroblastoma ATCC CRL-127 Yes <50% Kelly Human neuroblastoma ECACC 92110411 Yes >60% Kelly Human neuroblastoma DSMZ ACC 355 Yes >60% LA1-55n Human neuroblastoma ECACC 06041203 Yes >60% LA-N-1 Human neuroblastoma ECACC 06041201 -- <25% MC-IXC Human neuroepithelioma ATCC CRL-2270 -- <25% MHH-NB-11 Human neuroblastoma DSMZ ACC 157 No >60% N1E-115 Mouse neuroblastoma ATCC CCL-2263 Yes >60% N4TG3 Mouse neuroblastoma DSMZ ACC 101 No >60% N18TG2 Mouse neuroblastoma DSMZ ACC 103 No >60% NB4 1A3 Mouse neuroblastoma ECACC 89121405 No >60% ND3 Mouse neuroblastoma/primary ECACC 92090901 No >60% neonatal rat DRG hybrid ND7/23 Mouse neuroblastoma/primary rat ECACC 92090903 No >60% DRG hybrid ND8 Mouse neuroblastoma/primary ATCC No >60% neonatal rat DRG hybrid ND8/34 Mouse neuroblastoma ECACC 92090904 No >60% ND15 Mouse neuroblastoma/primary ECACC 92090907 No >60% neonatal rat DRG hybrid ND27 Mouse neuroblastoma/primary rat ECACC 92090912 No >60% DRG hybrid NB69 Human neuroblastoma ECACC 99072802 No >60% NDC Mouse neuroblastoma/primary ECACC 92090913 No >60% neonatal rat DRG hybrid Neuro-2a Mouse neuroblastoma ATCC CCL-131 Yes >60% NG108-15 Mouse neuroblastoma/rat glioma ECACC 88112302 Yes >60% hybrid NG115-401L Mouse neuroblastoma/rat glioma ECACC 87032003 No <50% hybrid NS20Y Mouse neuroblastoma DSMZ ACC 94 No >60% PC12 Rat pheochromocytoma ATCC CRL-1721 Yes >60% SH-SY5Y Human neuroblastoma ATCC CRL-2266 Yes >60% SiMa Human neuroblastoma DSMZ ACC 164 Yes >60% SK-N-BE(2)- Human neuroblastoma ATCC CRL-2271 Yes <50% C SK-N-AS Human neuroblastoma ATCC CRL-2137 No >60% SK-N-DZ Human neuroblastoma ATCC CRL-2149 No >60% SK-N-F1 Human neuroblastoma ATCC CRL-2142 No <50% SK-N-MC Human neuroblastoma ATCC HTB-10 -- <25% SK-N-SH Human neuroblastoma ECACC 86012802 No >60% TE 189.T Spinal cord ATCC CRL-7947 No >60%

Example II

Evaluation of Growth Conditions on Neurotoxin Uptake in Candidate Cell Lines

[0198] The following example illustrates how to determine growth conditions for established cell lines that maximize susceptible to BoNT/A intoxication or have BoNT/A uptake capacity.

1. Effects of Cell Differentiation on Neurotoxin Uptake of Candidate Cell Lines.

[0199] To determine whether cell differentiation improved neurotoxin uptake, cell lines exhibiting uptake of 1 nM BoNT/A were transferred into serum-free medium to induced differentiation. A suitable density of cells from a stock culture of the cell line being tested was plated into the wells of 24-well tissue culture plates containing 1 mL of a serum-free medium containing Minimum Essential Medium with 2 mM GlutaMAX.TM. I with Earle's salts, 0.1 mM Non-Essential Amino Acids, 10 mM HEPES, 1 mM Sodium Pyruvate, 100 units/mL Penicillin, and 100 .mu.g/mL Streptomycin. These cells were incubated in a 37.degree. C. incubator under 5% carbon dioxide until the cells differentiated, as assessed by standard and routine morphological criteria, such as growth arrest and neurite extension (approximately 2 to 3 days). As a control, a suitable density of cells from a stock culture of the cell line being tested was plated into the wells of 24-well tissue culture plates containing 1 mL of an appropriate growth medium (Table 1). These undifferentiated control cells were grown in a 37.degree. C. incubator under 5% carbon dioxide until cells reach the desired density (approximately 18 to 24 hours). The media from both differentiated and undifferentiated control cultures was aspirated from each well and replaced with fresh media containing either 0 (untreated sample), 0.1 nM, 0.3 nM, or 1 nM of a BoNT/A complex. After an overnight incubation, the cells were washed and harvested as described in Example I.

[0200] To detect for the presence of cleaved SNAP-25 products, an aliquot from each harvested sample was analyzed by Western blot as described in Example I, except that harvested samples are separated by SDS-PAGE using 12% 26-well Criterion gels (Bio-Rad Laboratories, Hercules, Calif.), and the rabbit polyclonal .alpha.-SNAP-25.sub.197 antibody serum was used as the primary antibody (see Example IV). Table 3 indicates the cell lines that exhibited a SNAP-25 cleavage product when treated with 0.1 nM BoNT/A. Of the cell lines tested, only the SiMa and Neuro-2a cell lines exhibited an uptake of 0.1 nM BoNT/A in the undifferentiated state. However, besides SiMa and Neuro-2a, the cell lines N18, LA1-55n, PC12, and SH-SY5Y all exhibited an uptake of 0.1 nM BoNT/A in the differentiated state.

TABLE-US-00003 TABLE 3 Effects of Cell Differentiation on Neurotoxin Uptake of Candidate Cell Lines. 0.1 nM BoNT/A Uptake Cell Line Description Source Undifferentiated Differentiated BE(2)-M17 Human neuroblastoma ATCC CRL-2267 No No Kelly Human neuroblastoma DSMZ ACC 355 No No LA1-55n Human neuroblastoma ECACC 06041203 No Yes N1E-115 Mouse neuroblastoma ATCC CCL-2263 No Not Tested N4TG3 Mouse neuroblastoma DSMZ ACC 101 No Not Tested N18 Mouse neuroblastoma/rat ECACC 88112301 No Yes glioma hybrid Neuro-2a Mouse neuroblastoma ATCC CCL-131 Yes Yes NG108-15 Mouse neuroblastoma/rat ECACC 88112302 No Not Tested glioma hybrid PC12 Rat pheochromocytoma ATCC CRL-1721 No Yes SH-SY5Y Human neuroblastoma ATCC CRL-2266 No Yes SiMa Human neuroblastoma DSMZ ACC 164 Yes Yes SK-N- Human neuroblastoma ATCC CRL-2271 No Not Tested BE(2)-C

2. Effects of Ganglioside Treatment on Neurotoxin Uptake of Differentiated Candidate Cell Lines.

[0201] To determine whether treatments improving low-affinity binding of neurotoxin could improve neurotoxin uptake, differentiated cell lines exhibiting uptake of 1 nM BoNT/A were treated with ganglioside GT1b. A suitable density of cells from a stock culture of the cell line being tested was plated into the wells of 24-well tissue culture plates containing serum-free medium as described above, with or without 25 .mu.g/mL GT1b (Alexis Biochemicals, San Diego, Calif.). These cells were incubated in a 37.degree. C. incubator under 5% carbon dioxide until the cells differentiated, as assessed by standard and routine morphological criteria as described above. The media was aspirated from each well and replaced with fresh serum-free media containing either 0 (untreated sample), 1.9 pM, 3.7 pM, 7.4 pM, 14.8 pM, 29.7 pM, 59.4 pM, 118.8 pM, 237.5 pM, 574 pM, 950 pM, and 1900 pM of a BoNT/A complex. The cell lines were incubated at two different times, 24 hours and 48 hours. After toxin incubation, the cells were washed and harvested as described in Example I.

[0202] To detect for the presence of cleaved SNAP-25 products, an aliquot from each harvested sample was analyzed by Western blot as described in Example I, except that harvested samples are separated by SDS-PAGE using 12% 26-well Criterion gels (Bio-Rad Laboratories, Hercules, Calif.), and the rabbit polyclonal .alpha.-SNAP-25.sub.197 antibody serum was used as the primary antibody (see Example IV). Table 4 indicates the effects of gangliosides treatment on the ability of differentiated cell lines to uptake BoNT/A. These results indicate the lowest concentration of BoNT/A that will produce a detectable band of SNAP-25 cleavage product in the Western blot.

TABLE-US-00004 TABLE 4 Effects of Ganglioside Treatment on Neurotoxin Uptake of Candidate Cell Lines. BoNT/A Uptake 24 Hour 48 Hour Cell Line Description Source Incubation Incubation BE(2)-M17 Human neuroblastoma ATCC CRL-2267 237.5 pM 118.8 pM Kelly Human neuroblastoma DSMZ ACC 355 Not Tested Not Tested LA1-55n Human neuroblastoma ECACC 15 pM 7.4 pM 06041203 N1E-115 Mouse neuroblastoma ATCC CCL-2263 Not Tested Not Tested N4TG3 Mouse neuroblastoma DSMZ ACC 101 Not Tested Not Tested N18 Mouse neuroblastoma/rat ECACC 14.8 pM 7.4 pM glioma hybrid 88112301 Neuro-2a Mouse neuroblastoma ATCC CCL-131 7.4 pM 7.4 pM NG108-15 Mouse neuroblastoma/rat ECACC Not Tested Not Tested glioma hybrid 88112302 PC12 Rat pheochromocytoma ATCC CRL-1721 7.4 pM 7.4 pM SH-SY5Y Human neuroblastoma ATCC CRL-2266 Not Tested Not Tested SiMa Human neuroblastoma DSMZ ACC 164 1.9 pM 1.9 pM SK-N-BE(2)- Human neuroblastoma ATCC CRL-2271 Not Tested Not Tested C

3. Development of Serum-Free Media with Cell Differentiating Properties that Enhanced Neurotoxin Uptake of Candidate Cell Lines.

[0203] To determine whether treatment improvements that induce cell differentiation could improve neurotoxin uptake, SiMa, Neuro-2a and PC12 cell lines were grown in various serum-free medium to induced differentiation. A suitable density of cells from a stock culture of the cell line being tested was plated into the wells of 24-well tissue culture plates containing 1 mL of various test serum-free medium. Parameters tested were 1) the effect of different basal media on BoNT/A uptake (MEM and RPMI 1649); 2) the effect of the presence or absence of neurotrophic factors on BoNT/A uptake (N2 supplement and B27 supplement); 3) the effect of the presence or absence of differentiation factors on BoNT/A uptake (retinoic acid and nerve growth factor); and 4) the effect of the presence or absence of serum on BoNT/A uptake (serum-free media and reduced serum media). As a control, a suitable density of cells from a stock culture of the cell line being tested was plated into the wells of 24-well tissue culture plates containing 1 mL of a control serum-free media (Minimum Essential Medium, 2 mM GlutaMAX.TM. I with Earle's salts, 0.1 mM Non-Essential Amino Acids, 10 mM HEPES, 1 mM Sodium Pyruvate, 100 units/mL Penicillin, and 100 .mu.g/mL Streptomycin). These cells were incubated in a 37.degree. C. incubator under 5% carbon dioxide until the cells differentiated, as assessed by standard and routine morphological criteria, such as growth arrest and neurite extension (approximately 2 to 3 days). The media was aspirated from each well and replaced with fresh serum-free media containing either 0 (untreated sample), 0.005 pM, 0.015 pM, 0.05 pM, 0.14 pM, 0.42 pM, 1.2 pM, 3.7 pM, 11 pM, 33 pM, 100 pM and 300 pM of a BoNT/A complex. In addition, the differentiated cells were treated with BoNT/A for 24 hrs followed by a media change and 48 hrs incubation in fresh media without toxin to allow for the accumulation of SNAP-25 cleavage product. The cells were then washed and harvested as described in Example I.

TABLE-US-00005 TABLE 5 Serum Free Media Used for Differentiating Cell Lines. Cell Line Test Serum Free Media Composition LA1-55n Minimum Essential Medium with 2 mM GlutaMAX .TM. I with Earle's salts, 0.1 mM Non-Essential Amino- Acids, 10 mM HEPES, 1 .times. N2 supplement, and 1 .times. B27 supplement Neuro-2a Minimum Essential Medium, 2 mM GlutaMAX .TM. I with Earle's salts, 1 .times. B27 supplement, 1 .times. N2 supplement, 0.1 mM Non-Essential Amino Acids, 10 mM HEPES PC12 RPMI 1640, 2 mM GlutaMAX .TM., 1 .times. B27 supplement, 1 .times. N2 supplement, 10 mM HEPES, 1 mM sodium pyruvate, 1% Penicillin-Streptomycin and 50 ng/mL Nerve Growth Factor SiMa Minimum Essential Medium, 2 mM GlutaMAX .TM. I with Earle's salts, 1 .times. B27 supplement, 1 .times. N2 supplement, 0.1 mM Non-Essential Amino Acids, 10 mM HEPES

[0204] To detect for the presence of a SNAP-25 cleavage product, an aliquot from each harvested sample was analyzed by Western blot as described in Example I, except that harvested samples are separated by SDS-PAGE using 12% 26-well Criterion gels (Bio-Rad Laboratories, Hercules, Calif.), and an .alpha.-SNAP-25 rabbit polyclonal antibody serum was used (see Example IV). The most optimized media determined for each cell line is shown in Table 5. Table 6 indicates the lowest amount of a SNAP-25 cleavage product detected when the cell lines were grown in this optimized serum-free medium. Use of the optimized serum-free medium resulted in the detection of BoNT/A activity signals with acceptable signal-to-noise ratios in LA1-55n, Neuro-2a, PC-12, and SiMa cell lines (FIG. 2). For example, optimized differentiation conditions resulted in a 5-fold increase in SNAP-25 cleavage product detection as compared to the control serum-free media for Neuro-2a and PC12 cells, and almost 50-fold for SiMa cells. In addition, a minimal signal to noise ratio of 3:1 for the lower asymptote and 10:1 for the upper asymptote is required to develop a robust assay amenable for validation. Wth the exception of LA-1-55n, all optimized cell lines provided a signal to noise ratio for the lower asymptote of at least 3:1 when the signal detected from the 1.2 pM dose was compared to the background signal of 0 pM BoNT/A (FIG. 2). In addition, all optimized cell lines provided a signal to noise ratio for the upper asymptote of at least 100:1 when the signal from the 300 pM dose was compared to the background signal of 0 pM BoNT/A (FIG. 2). These results indicate that any of these cell lines could be used to develop an immuno-based method for detecting BoNT/A activity as disclosed in the present specification because the assay was detecting the presence of pM amounts of BoNT/A.

TABLE-US-00006 TABLE 6 Effects of Optimized Serum-Free Media on Neurotoxin Uptake of Candidate Cell Lines. BoNT/A Uptake Control Optimized Serum-Free Serum-Free Cell Line Description Source Media Media BE(2)-M17 Human neuroblastoma ATCC CRL-2267 Not Tested Not Tested Kelly Human neuroblastoma DSMZ ACC 355 Not Tested Not Tested LA1-55n Human neuroblastoma ECACC 06041203 7.4 pM 3.7 pM N1E-115 Mouse neuroblastoma ATCC CCL-2263 Not Tested Not Tested N4TG3 Mouse neuroblastoma DSMZ ACC 101 Not Tested Not Tested N18 Mouse neuroblastoma/rat ECACC 88112301 Not Tested Not Tested glioma hybrid Neuro-2a Mouse neuroblastoma ATCC CCL-131 3.7 pM 0.8 pM NG108-15 Mouse neuroblastoma/rat ECACC 88112302 Not Tested Not Tested glioma hybrid PC12 Rat pheochromocytoma ATCC CRL-1721 2.0 pM 0.42 pM SH-SY5Y Human neuroblastoma ATCC CRL-2266 Not Tested Not Tested SiMa Human neuroblastoma DSMZ ACC 164 0.23 pM 0.005 pM SK-N-BE(2)- Human neuroblastoma ATCC CRL-2271 Not Tested Not Tested C

Example III

Development of .alpha.-SNAP-25 Monoclonal Antibodies that Selectively Bind a SNAP-25 Epitope Having a Free Carboxyl-Terminus at the P.sub.1 Residue of the BoNT/a Cleavage Site Scissile Bond

[0205] The following example illustrates how to make .alpha.-SNAP-25 monoclonal antibodies that can selectively bind to a SNAP-25 epitope having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond.

1. Generation of .alpha.-SNAP-25 Monoclonal Antibodies.

[0206] To develop monoclonal .alpha.-SNAP-25 antibodies that bind an epitope comprising a carboxyl-terminus at the P.sub.1 residue from the BoNT/A cleavage site scissile bond from a SNAP-25 cleavage product, the 13-residue peptide CDSNKTRIDEANQ.sub.COOH (SEQ ID NO: 38) was designed as a SNAP-25 cleavage product antigen. This peptide comprises a flexible linker region and a N-terminal Cysteine residue for conjugation to KLH and amino acids 186-197 of human SNAP-25 (SEQ ID NO: 5) with a carboxylated C-terminal glutamine (SEQ ID NO: 38). The generation of monoclonal antibodies to well-chosen, unique peptide sequences provides control over epitope specificity, allowing the identification of a particular subpopulation of protein among a pool of closely related isoforms. Blast searches revealed that this peptide has high homology only to SNAP-25 and almost no possible cross-reactivity with other proteins in neuronal cells. The sequence was also carefully scrutinized by utilizing computer algorithms to determine hydropathy index, protein surface probability, regions of flexibility, and favorable secondary structure, followed by proper orientation and presentation of the chosen peptide sequence. The peptide was synthesized and conjugated to Keyhole Limpet Hemocyanin (KLH) to increase immunogenicity. Six Balb/c mice were immunized with this peptide, and after three immunizations in about eight weeks, the mice were bled for testing. The blood was allowed to clot by incubating at 4.degree. C. for 60 minutes. The clotted blood was centrifuged at 10,000.times.g at 4.degree. C. for 10 minutes to pellet the cellular debris. The resulting serum sample was dispensed into 50 .mu.l aliquots and stored at -20.degree. C. until needed.

[0207] A similar strategy based on other SNAP-25 antigens disclosed in the present specification is used to develop .alpha.-SNAP-25 monoclonal antibodies that bind an epitope comprising a carboxyl-terminus at the P.sub.1 residue from the BoNT/A cleavage site scissile bond from a SNAP-25 cleavage product. For example, the SNAP-25 antigen of SEQ ID NO: 45 can be conjugated to KLH instead of the SNAP-25 antigen of SEQ ID NO: 38. As another example, the amino acids 186-197 of human SNAP-25 from the SNAP-25 antigen of SEQ ID NO: 38 can be replaced with SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, or SEQ ID NO: 44.

2. Screening for the Presence of .alpha.-SNAP-25 Monoclonal Antibodies.

[0208] To determine the presence of an .alpha.-SNAP-25 monoclonal antibody that can selectively bind to a SNAP-25 antigen having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond, comparative ELISA and cell-based cleavage assay were performed using the extracted mouse serum. For comparative ELISA, two fusion proteins were constructed: BirA-HisTag.RTM.-SNAP-25.sub.134-197 of SEQ ID NO: 48 and the BirA-HisTag.RTM.-SNAP-25.sub.134-206 of SEQ ID NO: 49. BirA-HisTag.RTM.-SNAP-25.sub.134-197 comprised a naturally-biotinylated 16 amino acid BirA peptide of SEQ ID NO: 50 amino-terminally linked to a SNAP-25 peptide comprising amino acids 134-197 of SEQ ID NO: 5. BirA-HisTag.RTM.-SNAP-25.sub.134-206 comprised a naturally-biotinylated 16 amino acid BirA peptide of SEQ ID NO: 50 amino-terminally linked to a SNAP-25 peptide comprising amino acids 134-206 of SEQ ID NO: 5. These two substrates were suspended in 1.times.PBS at a concentration of 10 .mu.g/mL BirA-HisTag.RTM.-SNAP-25.sub.134-197 and the BirA-HisTag.RTM.-SNAP-25.sub.134-296. The BirA-HisTag.RTM.-SNAP-25.sub.134-197 and the BirA-HisTag.RTM.-SNAP-25.sub.134-206 were coated onto separate plates by adding approximately 100 .mu.l of the appropriate Substrate Solution and incubating the plates at room temperature for one hour. Washed plates were incubated at 37.degree. C. for one hour in 0.5% BSA in 1.times.TBS containing a 1:10 to 1:100 dilution of an antibody-containing serum derived from one of the six immunized mice (Mouse 1, Mouse 2, Mouse 3, Mouse 4, Mouse 5, and Mouse 6). Primary antibody probed plates were washed four times for 5 minutes each time in 200 .mu.l TBS, 0.1% TWEEN-20.RTM. (polyoxyethylene (20) sorbitan monolaureate). Washed plates were incubated at 37.degree. C. for 1 hour in 1.times.TBS containing a 1:10,000 dilution of goat polyclonal anti-mouse IgG antibody conjugated to Horseradish peroxidase (Pierce Biotechnology, Rockford, Ill.) as a secondary antibody. Secondary antibody-probed plates were washed four times in 200 .mu.l TBS, 0.1% TWEEN-20.RTM. (polyoxyethylene (20) sorbitan monolaureate). Chromogenic detection of the labeled SNAP-25 products were visualized by chromogenic detection using ImmunoPure TMB substrate kit (Pierce Biotechnology, Rockford, Ill.). The development of a yellow color in the BirA-HisTag.RTM.-SNAP-25.sub.134-197 coated plates, but not the BirA-HisTag.RTM.-SNAP-25.sub.134-206 coated plates, indicated that the .alpha.-SNAP-25 antibody preferentially recognized the SNAP-25.sub.197 cleavage product. The resulted indicated that of the six mice used for immunization, three mice (Mouse 2, Mouse 3, and Mouse 4) had higher titers and more specificity towards a SNAP-25 antigen having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond.

[0209] These results were confirmed using an ELISA light chain activity assay. A 96-well Reacti-Bind Streptavidin coated plates (Pierce Biotechnology, Rockford, Ill.) were prepared by adding approximately 100 .mu.l of the following Substrate Solution: Rows A-C were coated with 100 .mu.L of BirA-HisTag.RTM.-SNAP-25.sub.134-197 at twelve different concentrations; Rows D-H were coated with 100 .mu.L of BirA-HisTag.RTM.-SNAP-25.sub.134-296 at 10 .mu.g/mL. The plates were washed by aspirating the Substrate Solution and rinsing each well three times with 200 .mu.l TBS, 0.1% TWEEN-20.RTM. (polyoxyethylene (20) sorbitan monolaureate). Dilutions of BoNT/A were pre-reduced at 37.degree. C. for 20 minutes in BoNT/A Incubation Buffer (50 mM HEPES, pH 7.4, 1% fetal bovine serum, 10 pM ZnCl.sub.2, 10 mM dithiothrietol) and 100 .mu.l of the pre-reduced BoNT/A was added to the substrate-coated plates and incubated at 37.degree. C. for 90 minutes. BoNT/A treated plates were washed by aspirating the BoNT/A Incubation Buffer and rinsing each plate three times with 200 .mu.l TBS, 0.1% TWEEN-20.RTM. (polyoxyethylene (20) sorbitan monolaureate). Washed plates were incubated at 37.degree. C. for one hour in 0.5% BSA in 1.times.TBS containing a 1:10 to 1:100 dilution of the antibody-containing serum being tested. Primary antibody probed plates were washed four times for 5 minutes each time in 200 .mu.l TBS, 0.1% TWEEN-20.RTM. (polyoxyethylene (20) sorbitan monolaureate). Washed plates were incubated at 37.degree. C. for 1 hour in 1.times.TBS containing a 1:10,000 dilution of goat polyclonal anti-mouse IgG antibody conjugated to Horseradish peroxidase (Pierce Biotechnology, Rockford, Ill.) as a secondary antibody. Secondary antibody-probed plates were washed four times in 200 .mu.l TBS, 0.1% TWEEN-20.RTM. (polyoxyethylene (20) sorbitan monolaureate). Chromogenic detection of the labeled SNAP-25 products were visualized by chromogenic detection using ImmunoPure TMB substrate kit (Pierce Biotechnology, Rockford, Ill.). The development of a yellow color, which correlated with the presence of the SNAP-25.sub.197 cleavage product was detected in BoNT/A treated samples, but not untreated controls, using antibody-containing serum derived from all six immunized mice (Mouse 1, Mouse 2, Mouse 3, Mouse 4, Mouse 5, and Mouse 6). Thus, the comparative ELISA analysis indicated that of the mice used for immunization, three mice (Mouse 2, Mouse 3, and Mouse 4) had higher titers and more specificity towards a SNAP-25 antigen having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond.

[0210] For cell-based cleavage assay, a suitable density of PC12 cells were plated into 60 mm.sup.2 tissue culture plates containing 3 mL of an appropriate serum medium (Table 1). The cells were grown in a 37.degree. C. incubator under 5% carbon dioxide until cells reached the appropriate density. A 500 .mu.L transfection solution was prepared by adding 250 .mu.L of OPTI-MEM Reduced Serum Medium containing 15 .mu.L of LipofectAmine 2000 (Invitrogen Inc., Carlsbad, Calif.) incubated at room temperature for 5 minutes to 250 .mu.L of OPTI-MEM Reduced Serum Medium containing 10 pg of a pQBI-25/GFP-BoNT/A-LC expression construct (SEQ ID NO: 51). The pQBI-25/GFP-BoNT/A-LC expression construct comprises a pQBI-25 expression vector (Qbiogene Inc., Carlsbad, Calif.) whose promoter elements are functionally linked to a polynucleotide encoding the GFP-BoNT/A light chain of SEQ ID NO: 52. This transfection mixture was incubated at room temperature for approximately 20 minutes. The media was replaced with fresh unsupplemented media and the 500 .mu.L transfection solution was added to the cells. The cells were then incubated in a 37.degree. C. incubator under 5% carbon dioxide for approximately 6 to 18 hours. The cells were washed and harvested as described in Example II. To detect for the presence of the cleaved SNAP-25.sub.197 product, an aliquot from each harvested sample was analyzed by Western blot as described in Example II, except that the primary antibody used was a 1:1,000 dilution of the antibody-containing serum and the secondary antibody used was a 1:20,000 of mouse .alpha.-IgG Horseradish Peroxidase (Pierce Biotechnology, Rockford, Ill.). A single band corresponding to the SNAP-25.sub.197 cleavage product was detected in BoNT/A treated samples, but not untreated controls, using antibody-containing serum derived from three mice (Mouse 2, Mouse 3, and Mouse 4). Thus, the cell-based cleavage assay indicated that of the mice used for immunization, three mice (Mouse 2, Mouse 3, and Mouse 4) had higher titers and more specificity towards a SNAP-25 antigen having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond.

3. Production of Hybridomas.

[0211] To make hybridomas producing .alpha.-SNAP-25 monoclonal antibodies that can selectively bind to a SNAP-25 antigen having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond, the spleen from Mouse 2 was harvested three days subsequent to a final "booster" immunization and the spleen cells were fused with myeloma cells P.sub.3-X63 Ag8.653 using standard hybridoma protocols. These cells were plated into five 96-well plates and hybrids were selected using HAT medium. Within 8-14 days after fusion, the first screening of the approximately 480 parent clones was carried out using comparative ELISA with the BirA-HisTag.RTM.-SNAP-25.sub.134-197 and the BirA-HisTag.RTM.-SNAP-25.sub.134-206 peptides coated in two separate plates. The comparative ELISA provided a quick screen method to identify hybridomas producing antibodies specific for the cleaved SNAP-25.sub.197. The top 18 clones were subjected to further screening using the cell-based cleavage assay described above and immunostaining of LC/A transfected cells. (Table 7).

TABLE-US-00007 TABLE 7 Analysis of Supernatants Containing .alpha.-SNAP-25 Monoclonal Antibody Comparative ELISA Cell-Based Assay Clone OD SNAP-25.sub.197 OD SNAP-25.sub.206 Ratio.sub.197/206 Ratio.sub.206/197 SNAP-25.sub.197 SNAP-25.sub.206 1D3 1.805 0.225 8.02 0.13 +++ - 1F12 0.365 0.093 3.92 0.25 - - 1G10 0.590 0.137 4.31 0.23 ++ - 1H1 0.335 0.121 2.77 0.36 - - 1H8 0.310 0.302 1.03 0.97 + - 2C9 0.139 0.274 0.51 1.97 - - 2E2 0.892 0.036 24.78 0.04 ++ - 2E4 0.228 0.069 3.30 0.30 + - 2F11 1.095 1.781 0.61 1.63 - - 3C1 1.268 0.053 23.92 0.04 ++ - 3C3 0.809 0.052 15.56 0.06 ++ - 3E1 0.086 0.155 0.55 1.80 0 - 3E8 2.048 0.053 38.64 0.03 +++ - 3G2 0.053 0.158 0.34 2.98 - - 4D1 0.106 0.218 0.49 2.06 - - 4G6 0.061 0.159 0.38 2.61 - - 5A5 0.251 0.106 2.37 0.42 + - 5F11 0.243 0.061 3.98 0.25 - -

[0212] Clones 1D3, 1G10, 2E2, 3C1, 3C3, and 3E8 were further cloned by limiting dilution because the conditioned media produced by these clones comprised .alpha.-SNAP-25 antibodies with a preferential binding specificity having a ratio.sub.197/206 of at least 4:1 for the SNAP-25.sub.197 cleavage product relative to the SNAP-25.sub.206 uncleaved substrate and detected the SNAP-25.sub.197-cleavage product using the cell-based cleavage assay and the immunostaining of PC12 cells transfected with GFP-LC/A. Similarly clones 2C9, 2F11, 3G2, 4D1 and 4G6 were further cloned by limiting dilution because the conditioned media produced by these clones comprised .alpha.-SNAP-25 antibodies with a preferential binding specificity having a ratio.sub.206/197 of at least 1.5:1 for the SNAP-25.sub.206 uncleaved substrate relative to the SNAP-25.sub.197 cleavage product and detected the SNAP-25.sub.206-uncleaved substrate using the cell-based cleavage assay. These single-cell derived clones were screened again using comparative ELISA, cell-based cleavage, and immunostaining to confirm their affinity and specificity, and the antibodies were isotyped using standard procedures. Ascites were produced from clones 1D3B8 (IgM.k), 1G10A12 (IgG3.k), 2C9B10 (IgG3.k), 2E2A6 (IgG3.k), 2F11B6 (IgM.k), 3C1A5 (IgG2a.k), and 3C3E2 (IgG2a.k). Clone 3E8 stopped producing antibodies during the cloning process and could not be further evaluated.

4. Evaluation of Binding Specificity of .alpha.-SNAP-25 Monoclonal Antibodies.

[0213] To evaluate binding specificity of an .alpha.-SNAP-25 monoclonal antibody that can selectively bind to a SNAP-25 antigen having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond, ascites from clones 1D3B8, 1G10A12, 2C9B10, 2E2A6, 2F11B6, 3C1A5, and 3C3E2 were used to detect SNAP-25 cleavage product using the cell-based activity assay, immunocytochemistry and immunoprecipitation.

[0214] For the cell-based activity assay, binding specificity was determined by analyzing the ability of .alpha.-SNAP-25 antibody-containing ascites to detect the uncleaved SNAP-25.sub.206 substrate and the cleaved SNAP-25.sub.197 product by Western blot analysis. A suitable density of PC12 cells were plated into 60 mm.sup.2 tissue culture plates containing 3 mL of an appropriate serum medium, grown in a 37.degree. C. incubator under 5% carbon dioxide until an appropriate cell density was reached, and transfected with the either a transfection solution lacking the pQBI-25/GFP-BoNT/A-LC expression construct (untransfected cells) or a transfection solution containing the pQBI-25/GFP-BoNT/A-LC expression construct (transfected cells) as described above. The cells were washed and harvested as described in Example I. To detect for the presence of both the uncleaved SNAP-25.sub.206 substrate and the cleaved SNAP-25.sub.197 product, an aliquot from each harvested sample was analyzed by Western blot as described in Example I, except that the primary antibody used was a 1:100 dilution of the .alpha.-SNAP-25 monoclonal antibody-containing ascites and the secondary antibody used was a 1:20,000 of .alpha.-mouse IgG conjugated to Horseradish Peroxidase (Pierce Biotechnology, Rockford, Ill.). In addition, three commercially available mouse .alpha.-SNAP-25 monoclonal antibodies were tested. SMI-81 (Sternberger Monoclonals Inc., Lutherville, Md.), an .alpha.-SNAP-25 antibody the manufacturer indicates detects both the uncleaved SNAP-25.sub.206 substrate and the cleaved SNAP-25.sub.197 product, was used at a 15,000 dilution according to the manufacturer's recommendations. MC-6050 (Research & Diagnostic Antibodies, Las Vegas, Nev.), an .alpha.-SNAP-25 antibody the manufacturer indicates detects both the uncleaved SNAP-25.sub.206 substrate and the cleaved SNAP-25.sub.197 product, was used at a 1:100 dilution according to the manufacturer's recommendations. MC-6053 (Research & Diagnostic Antibodies, Las Vegas, Nev.), an .alpha.-SNAP-25 antibody the manufacturer indicates detects only the cleaved SNAP-25.sub.197 product, was used at a 1:100 dilution according to the manufacturer's recommendations.

[0215] Table 8 indicates the .alpha.-SNAP-25 antibody-containing ascites that detected only the SNAP-25.sub.197 cleavage product. The cell-based cleavage assay indicated that ascites produced from clones 1D3B8, 2C9B10, 2E2A6, 3C1A5, and 3C3E2 synthesize an .alpha.-SNAP-25 monoclonal antibody having high binding specificity for the SNAP-25.sub.197 cleavage product that allows for the selective recognition of this cleavage product relative to the SNAP-25.sub.206 uncleaved substrate. Commercial antibody SMI-81 detected the SNAP-25.sub.206 uncleaved substrate, but only poorly recognized the SNAP-25.sub.197 cleavage product (Table 8). Surprisingly, commercial antibody MC-6050 only detected the SNAP-25.sub.206 uncleaved substrate, and failed to recognize the SNAP-25.sub.197 cleavage product (Table 8). Even more surprisingly, commercial antibody MC-6050 only detected the SNAP-25.sub.206 uncleaved substrate, and failed to recognize the SNAP-25.sub.197 cleavage product, even though the manufacturer advertises that this antibody selectively detects the SNAP-25.sub.197 cleavage product (Table 8). Thus, this analysis indicates that while 1D3B8, 2C9B10, 2E2A6, 3C1A5, and 3C3E2 exhibit suitable selectivity for the SNAP-25.sub.197 cleavage product, 1G10A12 and 2F11B6 do not. In addition, commercial antibodies SMI-81, MC-6050 and MC-6053 all are unsuitable for the immuno-based methods disclosed in the present application because all failed to selectivity detect the SNAP-25.sub.197 cleavage product.

[0216] For immunocytochemistry analysis, binding specificity was determined by analyzing the ability of .alpha.-SNAP-25 antibody-containing ascites to detect the uncleaved SNAP-25.sub.206 substrate and the cleaved SNAP-25.sub.197 product by immunostaining. See e.g., Ester Fernandez-Salas et al., Plasma Membrane Localization Signals in the Light Chain of Botulinum Neurotoxin, Proc. Natl. Acad. Sci., U.S.A. 101(9): 3208-3213 (2004). A suitable density of PC12 cells were plated, grown, and transfected with either a transfection solution lacking the pQBI-25/GFP-BoNT/A-LC expression construct (untransfected cells) or a transfection solution containing the pQBI-25/GFP-BoNT/A-LC expression construct (transfected cells) as described above. The cells were washed in 1.times.PBS and fixed in 5 mL of PAF at room temperature for 30 minutes. Fixed cells were washed in phosphate buffered saline, incubated in 5 mL of 0.5% Triton.RTM. X-100 (polyethylene glycol octylphenol ether) in 1.times.PBS, washed in 1.times.PBS, and permeabilized in 5 mL of methanol at -20.degree. C. for six minutes. Permeabilized cells were blocked in 5 mL of 100 mM glycine at room temperature for 30 minutes, washed in 1.times.PBS, and blocked in 5 mL of 0.5% BSA in 1.times.PBS at room temperature for 30 minutes. Blocked cells were washed in 1.times.PBS and incubated at room temperature for two hours in 0.5% BSA in 1.times.PBS containing a 1:10 dilution of an ascites from a clonal hybridoma cell line being tested. Primary antibody probed cells were washed three times for 5 minutes each time in 1.times.PBS. Washed cells were incubated at room temperature for 2 hours in 1.times.PBS containing a 1:200 dilution of goat polyclonal anti-mouse immunoglobulin G, heavy and light chains (IgG, H+L) antibody conjugated to ALEXA.RTM. FLUOR 568 (Invitrogen Inc., Carlsbad, Calif.) as a secondary antibody. Secondary antibody-probed cells were washed three times for 5 minutes each time in 1.times.PBS. Washed cells were prepared for microscopic examination by mounting in VECTASHIELD.RTM. Mounting Media (Vector Laboratories, Burlingame, Calif.) and coverslipped. Images of signal detection were obtained with a Leica confocal microscope using appropriate laser settings. Table 8 indicates that the .alpha.-SNAP-25 antibody-containing ascites that specifically detected the SNAP-25.sub.197-cleavage product. The immunocytochemistry analysis indicated that ascites produced from clones 1D3B8, 2C9B10, 2E2A6, 3C1A5, and 3C3E2 synthesize an .alpha.-SNAP-25 monoclonal antibody having high binding specificity for the SNAP-25.sub.197 cleavage product that allows for the preferential recognition of this cleavage product relative to the SNAP-25.sub.206 uncleaved substrate.

[0217] For immunoprecipitation analysis, binding specificity was determined by analyzing the ability of Protein A (HiTrap.TM. Protein A HP Columns, GE Healthcare, Amersham, Piscataway, N.J.), purified .alpha.-SNAP-25 monoclonal antibodies to precipitate the uncleaved SNAP-25.sub.206 substrate and the cleaved SNAP-25.sub.197 product. See e.g., Chapter 8 Storing and Purifying Antibodies, pp. 309-311, Harlow & Lane, supra, 1998a. A suitable density of PC12 cells were plated, grown, and transfected with either a transfection solution containing a pQBI-25/GFP expression construct (control cells; SEQ ID NO: 53) or a transfection solution containing the pQBI-25/GFP-BoNT/A-LC expression construct (experimental cells) as described above. The pQBI-25/GFP expression construct comprises an expression vector whose promoter elements are functionally linked to a polynucleotide encoding GFP of SEQ ID NO: 54. After an overnight incubation, the cells were washed by aspirating the growth media and rinsing each well with 200 .mu.l 1.times.PBS. To harvest the cells, the PBS was aspirated, the cells were lysed by adding an Immunoprecipitation Lysis Buffer comprising 50 mM HEPES, 150 mM NaCl, 1.5 mM MgCl.sub.2, 1 mM EGDT, 10% glycerol, 1% Triton.RTM. X-100 (polyethylene glycol octylphenol ether) and a 1.times. COMPLETE.TM. Protease inhibitor cocktail (Roche Applied Biosciences, Indianapolis, Ind.) and incubating at 4.degree. C. for one hour. The lysed cells were centrifuged at 3,000.times.g at 4.degree. C. for 10 minutes to remove cellular debris and the supernatant transferred to a clean tube and diluted to a protein concentration of approximately 1 mg/mL. Approximately 5 pg of purified monoclonal antibody was added to 0.5 mL of diluted supernatant and incubated at 4.degree. C. for two hours. After primary antibody incubation, approximately 50 .mu.l of immobilized Protein G (Pierce Biotechnology, Rockford, Ill.) was added to the diluted supernatant and incubated at 4.degree. C. for one hour. The incubated supernatant was washed three times for 30 minutes each time by adding 0.5 mL of Immunoprecipitation Lysis Buffer, centrifuging at 300.times.g at 4.degree. C. for one minute to pellet the immobilized Protein G, and decanting the supernatant. After washing, the pellet was resuspended in 30 .mu.l of 1.times.SDS Loading Buffer and the sample was heated to 95.degree. C. for 5 minutes. To detect for the presence of both the uncleaved SNAP-25.sub.206 substrate and the cleaved SNAP-25.sub.197 product, an aliquot from each harvested sample was analyzed by Western blot as described in Example I, except that the primary antibody used was a 1:1,000 dilution of the .alpha.-SNAP-25 polyclonal antibody serum (see Example IV) and the secondary antibody used was a 1:20,000 of rabbit .alpha.-IgG Horseradish Peroxidase (Pierce Biotechnology, Rockford, Ill.). Table 8 indicates the .alpha.-SNAP-25 antibody-containing ascites that specifically pulled down the SNAP-25.sub.197-cleavage product by immunoprecipitation analysis. The immunoprecipitation analysis indicated that ascites produced from clones 2E2A6 and 3C1A5 synthesize an .alpha.-SNAP-25 monoclonal antibody having high binding specificity for the SNAP-25.sub.197 cleavage product that allows for the preferential recognition of this cleavage product relative to the SNAP-25.sub.206 uncleaved substrate.

TABLE-US-00008 TABLE 8 Analysis of Clone Ascites Containing .alpha.-SNAP-25 Monoclonal Antibody Cell-Based Assay Immunocytochemistry Immunoprecipitation Clone SNAP-25.sub.197 SNAP-25.sub.206 SNAP-25.sub.197 SNAP-25.sub.206 SNAP-25.sub.197 SNAP-25.sub.206 1D3B8 ++ - ++ - Not Tested Not Tested 1G10A12 ++ ++ Not Tested Not Tested Not Tested Not Tested 2C9B10 ++ - ++ - Not Tested Not Tested 2E2A6 ++ - ++ - ++ - 2F11B6 + + + + Not Tested Not Tested 3C1A5 ++ - ++ - ++ - 3C3E2 + - Not Tested Not Tested Not Tested Not Tested MC-6050 - + Not Tested Not Tested Not Tested Not Tested MC-6053 - + Not Tested Not Tested Not Tested Not Tested SMI-81 -/+ ++ Not Tested Not Tested Not Tested Not Tested

5. Evaluation of Binding Affinity of .alpha.-SNAP-25 Monoclonal Antibodies.

[0218] To determine the binding affinity of an .alpha.-SNAP-25 monoclonal antibody showing high binding specificity for either the SNAP-25.sub.197 cleavage product or the SNAP-25.sub.206 uncleaved substrate, binding affinity assays were performed on a BIAcore.TM. 3000 instrument using carboxymethyl dextran (CM5) sensor chips (BIAcore, Inc., Piscataway, N.J.). Runs were conducted at 25.degree. C. with HBS-EP buffer comprising 10 mM HEPES (pH 7.4), 150 mM sodium chloride, 3 mM EDTA, 0.005% (v/v) surfactant P.sub.20 at a flow rate of 10 .mu.l/min. SNAP-25 peptides comprising amino acids 134-197 of SEQ ID NO: 5 (SNAP-25.sub.134-197) or amino acids 134-206 of SEQ ID NO: 5 (SNAP-25.sub.134-206) were covalently attached to the surface of the CM5 sensor chips using standard amine coupling. Briefly, the CM5 chips were activated by a 7 minute injection of a mixture of 0.2 M 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and 0.05 M N-hydroxysuccimide; the SNAP-25 peptides were then injected in 10 mM sodium acetate (pH 4.0) for 20 min at a flow rate of 10 .mu.L/min; and unreacted succimide esters were blocked by a 7-min injection of 1 M ethanolamine hydrochloride, pH 8.5. The immobilized amount of SNAP-25.sub.134-197 or SNAP-25.sub.134-206 on the chip was reflected by a 100-150 increase in response units (about 0.10-0.15 ng/mm.sup.2). Antibody samples comprising either ascites or purified monoclonal antibodies produced from clones 1D3B8, 2C9B10, 2E2A6, 3C1A5, and 3C3E2, as well as, commercially available .alpha.-SNAP-25 antibodies were passed over the surface of the CM5 chips allowing an association time of 10 min and a dissociation time of 20 min. The surfaces were regenerated between runs by a 1 minute injection of 10 mM glycine-HCl (pH 2.5) at a flow rate of 15 .mu.L/min. Sensorgram curves were fitted to a 1:1 kinetic binding model with the BIAevaluation.TM. 3.0 software.

[0219] The results indicate that both 2E2A6 and 3C1A5 were highly specific for cleaved SNAP-25.sub.197 product over SNAP-25 uncleaved substrate (Table 9). When compared to the binding affinities of MC-6050 and MC-6053, 1D3B6 had an approximately 10-fold higher equilibrium disassociation constant for the SNAP-25 cleavage product relative to these commercial antibodies (Table 9). Interestingly, 2E2A6 had only a slightly lower equilibrium disassociation constant for the SNAP-25 cleavage product relative to these commercial antibodies (0.405 nM versus 0.497 and 0.508)(Table 9). As neither of these commercial .alpha.-SNAP-25 antibodies selectively recognized the SNAP-25 cleavage product (Table 8), an equilibrium disassociation constant lower than about 0.5 nM appears, in part, critical to achieve such selectivity. Similarly, when compared to the binding affinities of MC-6050 and MC-6053, 2E2A6 had an about at least one-fold slower off rate/dissociation constant (6.74.times.10.sup.-5 versus 8.82.times.10.sup.-4 s.sup.-1 and 1.18.times.10.sup.-3 s.sup.-1) (Table 9). This further suggests that an off rate/dissociation constant lower than about 8.82.times.10.sup.-4 appears, in part, critical to achieve selective binding for the SNAP-25 cleavage product. This result is consistent with 1D3B8, which had an off rate/dissociation constant of 5.78.times.10.sup.-5 s.sup.-1 (Table 9).

TABLE-US-00009 TABLE 9 Analysis of Binding Affinity .alpha.-SNAP-25 Monoclonal Antibodies SPR 1D3B8 2E2A6* Parameter SNAP-25.sub.197 SNAP-25.sub.206.sup.a SNAP-25.sub.197 SNAP-25.sub.206.sup.b Ka (M.sup.-1 s.sup.-1) 1.06 .times. 10.sup.6 -- 1.70 .times. 10.sup.6 -- (1.66 .times. 10.sup.5).sup. (--) Kd (s.sup.-1) 5.78 .times. 10.sup.-5 -- 1.53 .times. 10.sup.-4 -- (6.74 .times. 10.sup.-5) (--) KD (nM) 0.050 -- 0.090 -- (0.405) (--) SPR 3C1A5 2C9B10 Parameter SNAP-25.sub.197 SNAP-25.sub.206.sup.c SNAP-25.sub.197 SNAP-25.sub.206.sup.d Ka (M.sup.-1 s.sup.-1) 2.17 .times. 10.sup.5 -- 1.15 .times. 10.sup.4 -- Kd (s.sup.-1) 2.88 .times. 10.sup.-4 -- 3.11 .times. 10.sup.-4 -- KD (nM) 1.33 -- 27.1 -- SPR MC-6050 MC-6053 Parameter SNAP-25.sub.197 SNAP-25.sub.206 SNAP-25.sub.197 SNAP-25.sub.206 Ka (M.sup.-1 s.sup.-1) 1.78 .times. 10.sup.6 3.06 .times. 10.sup.2 2.32 .times. 10.sup.6 1.06 .times. 10.sup.2 Kd (s.sup.-1) 8.82 .times. 10.sup.-4 6.07 .times. 10.sup.-3 1.18 .times. 10.sup.-3 2.56 .times. 10.sup.-5 KD (nM) 0.497 19,800 0.508 240 *Two independent runs were conducted for this antibody with two different chips. .sup.aNo binding was observed when up to 125 nM of .alpha.-SNAP-25 monoclonal antibody 1D3B8 was passed over the surface of the CM5 sensor chip after a 10 minute association time. .sup.bNo binding was observed when up to 10 .mu.M of .alpha.-SNAP-25 monoclonal antibody 2E2A6 was passed over the surface of the CM5 sensor chip after a 10 minute association time. .sup.CNo binding was observed when up to 100 nM of .alpha.-SNAP-25 monoclonal antibody 3C1A5 was passed over the surface of the CM5 sensor chip after a 10 minute association time. .sup.dNo binding was observed when up to 100 nM of .alpha.-SNAP-25 monoclonal antibody 2C9B10 was passed over the surface of the CM5 sensor chip after a 10 minute association time.

6. Sequencing of the Epitope from Isolated .alpha.-SNAP-25 Monoclonal Antibodies.

[0220] To determine the epitope of an isolated .alpha.-SNAP-25 monoclonal antibody that can selectively bind to a SNAP-25 antigen having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond, the polynucleotide molecule encoding the variable heavy (V.sub.H) and variable light (V.sub.L) chains of the .alpha.-SNAP-25 monoclonal antibody produced by hybridomas 1D3B8, 2C9B10, 2E2A6, 3C1A5 and 3C3E2 were sequenced. mRNA was extracted and purified from each hybridoma using standard protocols and reversed transcribed into cDNA using either an oligo dT anti-sense primer or a gene-specific (murine IgG1 CH and kappa CL) anti-sense primer. Specific murine and human constant domain primers were used to amplify the cDNA by PCR after cDNA production to determine the isotype of the antibody. Degenerate V.sub.H and V.sub.L primers were used to amplify the variable domains from the cDNA. For 5'RACE, a homopolymeric dCTP tail was added to the 3' end of the cDNA. The heavy and light chains were then amplified with an oligo dG sense primer and a gene specific (CH/KC) anti-sense primer. PCR products included the sequence of the signal peptide, variable domains and constant domains up to the anti-sense primer. The PCR products were gel purified to remove small fragments, and cloned into a blunt or TA vector for sequencing. Five independent clones for each chain were sequenced and alignments of V.sub.H and V.sub.L chains and consensus sequences were determined (Table 10). Methods used to determine the V.sub.H and V.sub.L amino acid sequences are described in, e.g., Roger A. Sabbadini, et al., Novel Bioactive Lipid Derivatives and Methods of Making and Using Same, U.S. Patent Publication 2007/0281320; and Peter Amersdorfer, et al., Molecular Characterization of Murine Humoral Immune Response to Botulinum Neurotoxin Type A Binding Domain as Assessed by Using Phage Antibody Libraries, 65(9) Infect. Immun. 3743-3752, each of which is hereby incorporated by reference in its entirety. In addition, commercial services are available to sequence the variable heavy (V.sub.H) and variable light (V.sub.L) chains of an antibody and identify the CDR regions, see, e.g., Fusion Antibodies Ltd., Northern Ireland.

[0221] The polynucleotide sequence comprising the V.sub.H and V.sub.L chains of the .alpha.-SNAP-25 monoclonal antibody produced by the hybridomas disclosed in the present specification is as follows: 1D3B8 V.sub.H (SEQ ID NO: 71), 2C9B10 V.sub.H (SEQ ID NO: 73), 2E2A6 V.sub.H (SEQ ID NO: 75), 3C1A5 V.sub.H variant 1 (SEQ ID NO: 77), 3C1A5 V.sub.H variant 2 (SEQ ID NO: 79), 3C3E2 V.sub.H (SEQ ID NO: 81); 1D3B8 V.sub.L (SEQ ID NO: 83), 2C9B10 V.sub.L (SEQ ID NO: 85), 2E2A6 V.sub.L (SEQ ID NO: 87), 3C1A5 V.sub.L (SEQ ID NO: 89), and 3C3E2 V.sub.L (SEQ ID NO: 91). The amino acid sequence comprising the V.sub.H and V.sub.L chains of the .alpha.-SNAP-25 monoclonal antibody produced by the hybridomas disclosed in the present specification is as follows: 1D3B8 V.sub.H (SEQ ID NO: 72), 2C9B10 V.sub.H (SEQ ID NO: 74), 2E2A6 V.sub.H (SEQ ID NO: 76), 3C1A5 V.sub.H variant 1 (SEQ ID NO: 78), 3C1A5 V.sub.H variant 2 (SEQ ID NO: 80), 3C3E2 V.sub.H (SEQ ID NO: 82); 1D3B8 V.sub.L (SEQ ID NO: 84), 2C9B10 V.sub.L (SEQ ID NO: 86), 2E2A6 V.sub.L (SEQ ID NO: 88), 3C1A5 V.sub.L (SEQ ID NO: 90), and 3C3E2 V.sub.L (SEQ ID NO: 92). The amino acid sequences comprising the V.sub.H and V.sub.L CDR domains of the .alpha.-SNAP-25 monoclonal antibody produced by the hybridomas 1D3B8, 2C9B10, 2E2A6, 3C1A5, and 3C3E2 are given in Table 10.

TABLE-US-00010 TABLE 10 CDR Sequences of V.sub.H and V.sub.L domains from .alpha.-SNAP-25 Monoclonal Antibodies SEQ CDR Sequence Identified In ID NO: V.sub.H CDR 1 TFTDHSIH 2E2A6 93 2C9B10 3C1A5 variant 2 V.sub.H CDR 1 TFTNYVIH 3C1A5 variant 1 94 3C3E2 V.sub.H CDR 1 IFTDHALH 1D3B8 95 V.sub.H CDR 2 YIFPGNGNIEYNDKFKG 2E2A6 96 V.sub.H CDR 2 YLFPGNGNFEYNEKFKG 2C9B10 97 3C1A5 variant 2 V.sub.H CDR 2 YINPYNDGSKYNEKFKG 3C1A5 variant 1 98 3C3E2 V.sub.H CDR 2 YIFPGNGNIEYNEKFKG 1D3B8 99 V.sub.H CDR 3 KRMGY 2E2A6 100 3C1A5 variant 2 V.sub.H CDR 3 KKMDY 2C9B10 101 1D3B8 V.sub.H CDR 3 ARHLANTYYYFDY 3C1A5 variant 1 102 3C3E2 V.sub.L CDR 1 RSSQSIVHSNGNTYLE 1D3B8 103 V.sub.L CDR 1 RTTENIYSYFV 2C9B10 104 V.sub.L CDR 1 RASKSVSTSGYSYMH 2E2A6 105 V.sub.L CDR 1 KASQDIKSYLS 3C1A5 106 V.sub.L CDR 1 RASQNIGNYLH 3C3E2 107 V.sub.L CDR 2 KVSNRFS 1D3B8 108 V.sub.L CDR 2 NAKSLAE 2C9B10 109 V.sub.L CDR 2 LVSNLES 2E2A6 110 V.sub.L CDR 2 YATSLAD 3C1A5 111 V.sub.L CDR 2 YASQSIS 3C3E2 112 V.sub.L CDR 3 FQGSHVPPT 1D3B8 113 V.sub.L CDR 3 QHHYGTPYT 2C9B10 114 V.sub.L CDR 3 QHIRELTRS 2E2A6 115 V.sub.L CDR 3 LQHGESPFT 3C1A5 116 V.sub.L CDR 3 QQSDTWPLT 3C3E2 117

[0222] Non-limiting examples of amino acid sequences comprising V.sub.H CDR domain variants of the .alpha.-SNAP-25 monoclonal antibody produced by the hybridomas disclosed in the present specification include V.sub.H CDR1 variant SEQ ID NO: 118 for 1D3B8; V.sub.H CDR1 variant SEQ ID NO: 119 for 2C9B10, 2E2A6 and 3C1A5 V.sub.H variant 2; V.sub.H CDR1 variant SEQ ID NO: 120 for 3C1A5 V.sub.H variant 1 and 3C3E2; V.sub.H CDR2 variant SEQ ID NO: 121 for 1D3B8 and 2E2A6; V.sub.H CDR2 variant SEQ ID NO: 122 for 2C9B10 and 3C1A5 V.sub.H variant 2; V.sub.H CDR2 variant SEQ ID NO: 123 for 3C1A5 V.sub.H variant 1, and 3C3E2; V.sub.H CDR3 variant MDY for 1D3B8 and 2C9B10; V.sub.H CDR3 variant MGY for 2E2A6 and 3C1A5 V.sub.H variant 2; and V.sub.H CDR3 variant SEQ ID NO: 124 for 3C1A5 V.sub.H variant 1 and 3C3E2. Non-limiting examples of amino acid sequences comprising V.sub.L CDR domain variants of the .alpha.-SNAP-25 monoclonal antibody produced by the hybridomas disclosed in the present specification include V.sub.L CDR1 variant SEQ ID NO: 125 for 1D3B8; V.sub.L CDR1 variant SEQ ID NO: 126 for 2C9B10; V.sub.L CDR1 variant SEQ ID NO: 127 for 2E2A6; V.sub.L CDR1 variant SEQ ID NO: 128 for 3C1A5; V.sub.L CDR1 variant SEQ ID NO: 129 for 3C3E2; V.sub.L CDR2 variant KVS for 1D3B8; V.sub.L CDR2 variant NAK for 2C9B10; V.sub.L CDR2 variant LVS for 2E2A6; V.sub.L CDR2 variant YAT for 3C1A5; and V.sub.L CDR2 variant YAS for 3C3E2.

Example IV

Development of .alpha.-SNAP-25 Polyclonal Antibodies that Selectively Bind a SNAP-25 Epitope Having a Free Carboxyl-Terminus at the P.sub.1 Residue of the BoNT/a Cleavage Site Scissile Bond

[0223] The following example illustrates how to make .alpha.-SNAP-25 polyclonal antibodies that can selectively bind to a SNAP-25 epitope having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond.

[0224] To develop .alpha.-SNAP-25 polyclonal antibodies that bind an epitope comprising a carboxyl-terminus at the P1 residue from the BoNT/A cleavage site scissile bond from a SNAP-25 cleavage product, the 10-residue peptide CGGGRIDEANQ (SEQ ID NO: 46) was designed as a SNAP-25 cleavage product antigen. This peptide comprising a N-terminal Cysteine residue for conjugation to KLH, a G-spacer flexible spacer (GGG) linked to amino acids 191-197 of human SNAP-25 (SEQ ID NO: 5) and has a carboxylated C-terminal glutamine. Blast searches revealed that this peptide has high homology only to SNAP-25 and almost no possible cross-reactivity with other proteins in neuronal cells. The sequence was also carefully scrutinized by utilizing computer algorithms to determine hydropathy index, protein surface probability, regions of flexibility, and favorable secondary structure, followed by proper orientation and presentation of the chosen peptide sequence. The peptide was synthesized and conjugated to Keyhole Limpet Hemocyanin (KLH) to increase immunogenicity. Before the animals were immunized, naive rabbits were first screened against cell lysates from candidate cell lines in a Western blot in order to identify animals that had no immunoreactivity to the proteins present in the cell lysates. Two pre-screened rabbits were immunized with this peptide, and after three immunizations in about eight weeks, the rabbits were bled for testing. The blood was allowed to clot by incubating at 4.degree. C. for 60 minutes. The clotted blood was centrifuged at 10,000.times.g at 4.degree. C. for 10 minutes to pellet the cellular debris. The resulting serum sample was dispensed into 50 .mu.L aliquots and stored at -20.degree. C. until needed.

[0225] A similar strategy based on other SNAP-25 antigens disclosed in the present specification is used to develop .alpha.-SNAP-25 polyclonal antibodies that bind an epitope comprising a carboxyl-terminus at the P.sub.1 residue from the BoNT/A cleavage site scissile bond from a SNAP-25 cleavage product. For example, the SNAP-25 antigen of SEQ ID NO: 47 can be conjugated to KLH instead of the SNAP-25 antigen of SEQ ID NO: 46. As another example, the amino acids 191-197 of human SNAP-25 from the SNAP-25 antigen of SEQ ID NO: 38 can be replaced with SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 147 or SEQ ID NO: 148.

2. Screening for the Presence of .alpha.-SNAP-25 Polyclonal Antibodies.

[0226] To determine the presence of .alpha.-SNAP-25 polyclonal antibodies that can selectively bind to a SNAP-25 antigen having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond, comparative ELISA and cell-based cleavage assays were performed using the extracted rabbit serum as described in Example III. The serum from both rabbits contained .alpha.-SNAP-25 polyclonal antibodies that can selectively bind to a SNAP-25 antigen having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond. The .alpha.-SNAP-25 rabbit polyclonal antibodies were designated as NTP 22 and NTP 23.

3. Purification of .alpha.-SNAP-25 Polyclonal Antibodies.

[0227] To purify .alpha.-SNAP-25 polyclonal antibodies that can selectively bind to a SNAP-25 antigen having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond, NTP 22 and NTP 23 antibodies from rabbit serum were purified using affinity columns containing the SNAP-25 antigen of SEQ ID NO: 46.

4. Evaluation of Binding Specificity of .alpha.-SNAP-25 Polyclonal Antibodies.

[0228] To evaluate binding specificity of an .alpha.-SNAP-25 polyclonal antibody that can selectively bind to a SNAP-25 antigen having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond, purified NTP 22 and NTP 23 .alpha.-SNAP-25 polyclonal antibodies were used to detect cleavage product using the cell-based activity assay, immunnocytochemistry and immunoprecipitation as described in Example III. The cell-based cleavage assay, immunocytochemistry analysis and immunoprecipitation analysis all indicated that NTP 22 and NTP 23 .alpha.-SNAP-25 polyclonal antibodies did not cross-react with uncleaved SNAP-25. Thus both NTP 22 and NTP 23 have high binding specificity for the SNAP-25.sub.197 cleavage product that allows for the preferential recognition of this cleavage product relative to the SNAP-25.sub.206 uncleaved substrate. Affinity for the antigens can be determined using SPR in the BiAcore as described in Example III.

Example V

Component and Condition Preparation for a Sandwich ELISA

[0229] The following example illustrates how to identify and prepare the components and conditions necessary to perform a sandwich ELISA useful for conducting immuno-based methods of detecting BoNT/A activity by detecting a SNAP-25 cleavage product using a .alpha.-SNAP-25 monoclonal antibody specific for a SNAP-25 having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond.

1. Preparation of Cell Lysates from Cells Treated with BoNT/A.

[0230] To obtain a BoNT/A treated cell lysate for analysis, a suitable density of cells from a stock culture of Neuro-2a was seeded into a T175 flask containing 50 mL of a serum-free medium containing Minimum Essential Medium, 2 mM GlutaMAX.TM. I with Earle's salts, 1.times.B27 supplement, 1.times.N2 supplement, 0.1 mM Non-Essential Amino Acids, 10 mM HEPES. These cells were incubated in a 37.degree. C. incubator under 5% carbon dioxide until the cells differentiated, as assessed by standard and routine morphological criteria, such as growth arrest and neurite extension (approximately 2 to 3 days). As a control, a suitable density of cells from a stock culture of Neuro-2a was seeded into a T175 flask containing 50 mL of an appropriate growth medium (Table 1). These undifferentiated control cells were grown in a 37.degree. C. incubator under 5% carbon dioxide until 50% confluence was reached (approximately 18 hours). The media from both differentiated and undifferentiated control cultures was aspirated from each well and replaced with fresh media containing either 0 (untreated sample) or 10 nM of a BoNT/A complex. After an overnight incubation, the cells were washed and the cells harvested by lysing in freshly prepared Triton X-100 Lysis Buffer (50 mM HEPES, 150 mM NaCl, 1.5 mM MgCl.sub.2, 1 mM EGTA, 1% Triton X-100) at 4.degree. C. for 30 minutes with constant agitation. Lysed cells were centrifuged at 4000 rpm for 20 min at 4.degree. C. to eliminate debris using a bench-top centrifuge. The protein concentrations of cell lysates were measured by Bradford assay.

2. Preparation and Identification of Sandwich ELISA Components.

[0231] To identify an appropriate capture antibody-detection antibody pair an ECL sandwich ELISA analysis was conducted on twenty-six different combinations of capture and detection antibody pairs comprising eleven different .alpha.-SNAP-25 capture antibodies and seven different .alpha.-SNAP-25 detection antibodies (Table 12). The .alpha.-SNAP-25 antibodies used were 2E2A6 and 3C1A5 .alpha.-SNAP-25 mouse monoclonal antibodies disclosed in the present specification, SMI-81, MC-6050, and MC-6053 .alpha.-SNAP-25 mouse monoclonal antibodies disclosed in the present specification, NTP 23 .alpha.-SNAP-25 rabbit polyclonal antibodies disclosed in the present specification, S9684 .alpha.-SNAP-25 rabbit polyclonal antibodies (Sigma, St. Louis, Mo.), H-50 .alpha.-SNAP-25 rabbit polyclonal antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.), C-18 .alpha.-SNAP-25 goat polyclonal antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.), N-19 .alpha.-SNAP-25 goat polyclonal antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.), and SP12 .alpha.-SNAP-25 mouse polyclonal antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.).

[0232] To prepare the capture antibody solution, the .alpha.-SNAP-25 monoclonal antibodies contained in the ascites from hybridoma cell lines 2E2A6 and 3C1A5 as well as the .alpha.-SNAP-25 MC-6050 and MC-6053 monoclonal antibodies were purified using a standard Protein A purification protocol. All other .alpha.-SNAP-25 antibodies were purchased as purified antibodies.

[0233] To prepare the detection antibody solution, the appropriate .alpha.-SNAP-25 antibody was conjugated to Ruthenium(II)-tris-bipyridine-(4-methysulfonate) NHS ester labeling reagent (Meso Scale Discovery, Gaithersburg, Md.) according to the manufacturer's instructions (Meso Scale Discovery, Gaithersburg, Md.). The conjugation reaction was performed by adding 30 .mu.L of distilled water reconstituted MSD SULFO-TAG.TM. stock solution to 200 .mu.L of 2 mg/mL .alpha.-SNAP-25 polyclonal antibodies and incubating the reaction at room temperature for 2 hours in the dark. The labeled antibodies were purified using a standard spin column protocol and the protein concentration determined using a standard colorimetric protein assay. The absorbance of the .alpha.-SNAP-25 antibody/MSD SULFO-TAG.TM. conjugate was measured at 455 nm using a spectrophotometer to determine the concentration in moles per liter. The detection antibody solution was stored at 4.degree. C. until needed.

[0234] To prepare the solid phase support comprising the capture antibody that is specific for a SNAP-25 cleavage product, approximately 5 .mu.L of the appropriate .alpha.-SNAP-25 monoclonal antibody solution (20 .mu.g/mL in 1.times.PBS) is added to each well of a 96-well MSD High Bind plate and the solution is allowed to air dry in a biological safety cabinet for 2-3 hours in order to liquid evaporate the solution. The capture antibody-bound wells were then blocked by adding 150 .mu.L of Blocking Buffer comprising 2% Amersham Blocking Reagent (GE Life Sciences, Piscataway, N.J.) and 10% goat serum (VWR, West Chester, Pa.) at room temperature for 2 hours. Blocked plates were sealed and stored at 4.degree. C. until needed.

[0235] To detect the presence of a cleaved SNAP-25 cleavage product by ECL sandwich ELISA analysis, the Blocking Buffer from stored plates was aspirated from the wells, 25 .mu.L of a lysate from cells treated with BoNT/A, as described above, was added to each well and the plates were incubated at 4.degree. C. for overnight. Plate wells were washed three times by aspirating the cell lysate and rinsing each well three times with 200 .mu.L 1.times.PBS, 0.1% TWEEN-20.RTM. (polyoxyethylene (20) sorbitan monolaureate). After washing, 25 .mu.l of 5 .mu.g/mL detection antibody solution comprising 2% Amersham Blocking Reagent in 1.times.PBS, 0.1% TWEEN-20.RTM. (polyoxyethylene (20) sorbitan monolaureate) was added to each well, the plate was sealed, and the sealed plate was incubated at room temperature at room temperature for 1 hour with shaking. After detection antibody incubation, the wells were washed three times with 200 .mu.L 1.times.PBS, 0.1% TWEEN-20.RTM. (polyoxyethylene (20) sorbitan monolaureate). After washing 150 .mu.L of 1.times.Read Buffer (Meso Scale Discovery, Gaithersburg, Md.) was added to each well and the plates were read using a SECTOR.TM. Imager 6000 Image Reader (Meso Scale Discovery, Gaithersburg, Md.). A ratio was calculated by dividing the signal obtained at the 10 nM dose for each antibody-pair by the signal obtained at the 0 nM dose for each antibody-pair (Table 12). These results indicated that among the twenty-six different combinations of antibody pairs tested, only three antibody pairs had signal-to-noise ratios above 10:1 for the higher dose tested: Pair No. 1 (2E2A6 mouse mAb and S9684 rabbit pAb), Pair No. 4 (3C1A5 mouse mAb and S9684 rabbit pAb), and Pair No. 18 (S9684 rabbit pAb and 2E2A6 mouse mAb). Antibody Pair 1 was chosen for further assay development.

TABLE-US-00011 TABLE 12 Screening of .alpha.-SNAP-25 Antibody Combinations Detection Detection Signal/Noise SNAP-25 SNAP-25 Ratio Antibody Capture Detection cleavage uncleaved (10 nm/0 Pair No. Antibody Antibody product substrate nM) 1 2E2A6 mouse S9684 rabbit pAb Yes No 26.6:1 mAb 2 2E2A6 mouse N-19 goat pAb Yes No 7.3:1 mAb 3 2E2A6 mouse H-50 rabbit pAb Yes No 0.9:1 mAb 4 3C1A5 mouse S9684 rabbit pAb Yes No 12.1:1 mAb 5 3C1A5 mouse N-19 goat pAb Yes No 1.9:1 mAb 6 3C1A5 mouse H-50 rabbit pAb Yes No 0.9:1 mAb 7 C-18 goat pAb S9684 rabbit pAb No No 0.8:1 8 C-18 goat pAb N-19 goat pAb No No 0.9:1 9 C-18 goat pAb H-50 rabbit pAb No No 0.9:1 10 H-50 rabbit pAb 2E2A6 mouse Yes No 0.9:1 mAb 11 H-50 rabbit pAb C-18 goat pAb No No 1.0:1 12 N-19 goat pAb 2E2A6 mouse Yes No 0.9:1 mAb 13 N-19 goat pAb C-18 goat pAb No No 1.1:1 14 NTP 23 rabbit pAb N-19 goat pAb Yes No 1.2:1 15 NTP 23 rabbit pAb C-18 goat pAb No No 1.1:1 16 NTP 23 rabbit pAb SP12 mouse pAb Yes No 1.3:1 17 NTP 23 rabbit pAb H-50 rabbit pAb Yes No 1.1:1 18 S9684 rabbit pAb 2E2A6 mouse Yes No 21.3:1 mAb 19 S9684 rabbit pAb C-18 goat pAb No No 0.7:1 20 S9684 rabbit pAb SMI-81mouse Yes Yes 1.2:1 mAb 21 SMI-81 mouse S9684 rabbit pAb Yes Yes 1.1:1 mAb 22 SMI-81 mouse N-19 goat pAb Yes Yes 1.0:1 mAb 23 SMI-81 mouse C-18 goat pAb No No 0.8:1 mAb 24 SP12 mouse pAb C-18 goat pAb No No 1.0:1 25 MC-6050 mouse S9684 rabbit pAb Yes Yes 5.0:1 mAb 26 MC-6053 mouse S9684 rabbit pAb Yes Yes 7.1:1 mAb

3. Optimization of Cell Differentiation Conditions.

[0236] To determine the optimal differentiation condition for a cell line comprising cells susceptible to BoNT/A intoxication when using a sandwich ELISA detection system, both various cell culture media and length of differentiation time were tested.

[0237] To determine an optimal differentiation medium, a suitable density of cells from a SiMa cell line was plated into the wells of Collagen IV coated 24-well cell culture plates containing 1 mL of one of the following medias and differentiation supplements: 1) RPMI 1640, 10% fetal bovine serum, 1% Penicillin-Streptomycin, 2 mM L-Glutamine, and 25 .mu.g/mL GT1b); 2) RPMI-1640, 1.times.B27 supplement, 1.times.N2 supplement, and 25 .mu.g/mL GT1b; 3) Minimum Essential Medium, 1.times.B27 supplement, 1.times.N2 supplement, and 25 .mu.g/mL GT1b; and 4) RPMI-1640, 10% BSA, 1.times.N2 supplement, 1.times.NGF supplement, and 25 .mu.g/mL GT1b. Cells were incubated in a 37.degree. C. incubator under 5% carbon dioxide until the cells differentiated, as assessed by standard and routine morphological criteria, such as growth arrest and neurite extension (approximately 3 days). The media was aspirated from each well and replaced with fresh media containing either 0 (untreated sample), 0.2 pM, 2 pM, or 20 pM of a BoNT/A complex. After an overnight treatment, the cells were washed, incubated for an additional two days without toxin to allow for the cleavage of the SNAP-25 substrate, and harvested as described above in Section 1. The protein concentrations of cell lysates were measured by Bradford assay. Detection of the presence of cleaved SNAP-25 product by ECL sandwich ELISA analysis was performed as described above using Antibody Pair 1. As discussed in Example I, undifferentiated cells did not take up toxin as effectively as differentiated cells. The most effective differentiation medium for increasing BoNT/A uptake and consequently SNAP-25 cleavage medium 3 (MEM+N2+B27), followed by medium 2 (RPMI-1640+N2+B27), and medium 4 (RPMI-1640+N2+NGF+BSA) (FIG. 3). Cells cultured in medium 2 resulted in more cleavage of the SNAP-25 as compared to the other media.

[0238] To determine an optimal differentiation time, a suitable density of cells from a SiMa cell line was plated into the wells of poly-D-lysine coated 96-well cell culture plates containing 100 .mu.L of a serum-free medium containing Minimum Essential Medium, 2 mM GlutaMAX.TM. I with Earle's salts, 1.times.B27 supplement, 1.times.N2 supplement, 0.1 mM Non-Essential Amino Acids, 10 mM HEPES and 25 .mu.g/mL GT1b. Cells were plated at four different days to obtain a differentiation time course testing 6 hrs, 24 h, 48 hrs, and 72 hrs, and were incubated in a 37.degree. C. incubator under 5% carbon dioxide The media was aspirated from each well and replaced with fresh media containing either 0 (untreated sample), 0.1 pM, 0.2 pM, 0.4 pM, 0.8 pM, 1.6 pM, 3.1 pM, 6.25 pM, 12.5 pM, or 25 pM of a BoNT/A complex. After an overnight treatment, the cells were washed, incubated for an additional two days without toxin to allow for the cleavage of the SNAP-25 substrate, and harvested as described above in Section 1. After harvesting, the protein concentrations of cell lysates and detection of the presence of cleaved SNAP-25 product by ECL sandwich ELISA analysis were performed as described above. The raw data obtained from the ECL imager was then transferred to SigmaPlot v. 9.0 and a 4-parameter logistics fit was used to define the dose-response curves. There were no constraints used for the 4-parameter logistic function when plotting the data. Graphical reports were generated using the following analysis: R2 (correlation coefficient), a (Max for data set), b (hillslope), and X0.+-.SE (EC.sub.50 value.+-.standard error). The results indicated that EC.sub.50 values of less than 2 pM could be achieved with cells differentiated for 48-72 hrs (FIG. 4). The finding that differentiated cells could be used between 48 hrs to 72 hrs of differentiation, with no significant changes on the performance of the cells, highlights the robustness of the assay. Although differentiation time periods less than 48 hrs may not be suitable for picomolar testing of formulated product, these lesser differentiation times are sensitive enough for bulk drug substance testing.

4. Optimization of BoNT/a Treatment Time.

[0239] To determine the optimal length of time cells form a cell line need to be treated with a BoNT/A, various lengths of BoNT/A treatment times were tested. A suitable density of cells from a SiMa cell line was plated into the wells of poly-D-lysine coated 96-well cell culture plates containing 100 .mu.L of a serum-free medium containing Minimum Essential Medium, 2 mM GlutaMAX.TM. I with Earle's salts, 1.times.B27 supplement, 1.times.N2 supplement, 0.1 mM Non-Essential Amino Acids, 10 mM HEPES and 25 .mu.g/mL GT1b. Cells were plated and incubated in a 37.degree. C. incubator under 5% carbon dioxide until the cells differentiated, as assessed by standard and routine morphological criteria, such as growth arrest and neurite extension (approximately 3 days). The media was aspirated from each well and replaced with fresh media containing either 0 (untreated sample), 0.1 pM, 0.2 pM, 0.4 pM, 0.8 pM, 1.6 pM, 3.1 pM, 6.3 pM, 12.5 pM, or 25 pM of a BoNT/A complex in RPMI 1640 growth medium in triplicate to generate a full dose-response. Five different BoNT/A treatment length regimens were performed: 1) a 6 hrs BoNT/A treatment, removal and washing of cells, an incubation of cells for 18 hr without BoNT/A, and harvesting of cells as described above in Section 1; 2) a 24 hrs BoNT/A treatment, removal and washing of cells, and harvesting of cells as described above in Section 1; 3) a 24 hrs BoNT/A treatment, removal and washing of cells, an incubation of cells for 24 hr without BoNT/A, and harvesting of cells as described above in Section 1; 4) a 24 hrs BoNT/A incubation, removal and washing of cells, an incubation of cells for 48 hr without BoNT/A, and harvesting of cells as described above in Section 1; and 5) a 24 hrs BoNT/A incubation, removal and washing of cells, an incubation of cells for 72 hr without BoNT/A, and harvesting of cells as described above in Section 1. After harvesting, the protein concentrations of cell lysates, detection of SNAP-25 cleavage product by ECL sandwich ELISA performed, and the EC.sub.50 calculated as described above. The results indicate that EC.sub.50 values of less than 2 pM could be achieved with any of the BoNT/A treatments tested (FIG. 5). Interestingly, the 24 hrs+24 hrs, 24 hrs+48 hrs, and 24 hrs+73 hrs BoNT/A treatment regimes generated essentially the same EC.sub.50 values, 1.0 pM, 1.1, pM and 0.9 pM respectively. The EC.sub.50 values generated for the 6 hrs+18 hrs and 24 hrs+0 hrs BoNT/A treatment regimes were 1.7 pM and 1.6 pM respectively. Although the amount of signal obtained was lower, these results indicate that BoNT/A treatment times between 6 hrs to 24 hrs plus one day to three days post-treatment incubation can be used to generate an EC.sub.50 that is adequate for detecting BoNT/A activity and give flexibility in the assay's overall time course.

5. Sensitivity of Immuno-Based Method of Detecting BoNT/a Activity.

[0240] To evaluate the sensitivity of the immuno-based methods of detecting BoNT/A activity disclosed in the present specification, the timing of BoNT/A uptake by cells susceptible to BoNT/A intoxication was determined. A suitable density of cells from a SiMa cell line was plated into the wells of poly-D-lysine coated 96-well cell culture plates containing 100 .mu.L of a serum-free medium containing Minimum Essential Medium, 2 mM GlutaMAX.TM. I with Earle's salts, 1.times.B27 supplement, 1.times.N2 supplement, 0.1 mM Non-Essential Amino Acids, 10 mM HEPES and 20 .mu.g/mL GT1b. Cells were incubated in a 37.degree. C. incubator under 5% carbon dioxide until the cells differentiated, as assessed by standard and routine morphological criteria, such as growth arrest and neurite extension (approximately 3 days). The media was aspirated from each well, replaced with fresh media containing 1 nM of a BoNT/A complex, and the BoNT/A treated cells were incubated at six different time points of 0 min (neurotoxin added and then immediately removed), 5 min, 10 min, 20 min, 30 min, and 60 min. A negative control of media with no BoNT/A (0 nM) was used. After incubation, the cells were washed and harvested as described above in Section 1. After harvesting, the protein concentrations of cell lysates, detection of SNAP-25 cleavage product by ECL sandwich ELISA performed, and the EC.sub.50 calculated as described above. The results indicated that uptake of BoNT/A by the cells took less than one minute before producing significant amounts of SNAP-25 cleavage product over background (FIG. 6).

6. Specificity of Immuno-Based Method of Detecting BoNT/a Activity.

[0241] To evaluate the specificity of the immuno-based methods of detecting BoNT/A activity disclosed in the present specification, the capacity of cells susceptible to BoNT/A intoxication to accurately distinguish BoNT/A to the exclusion of partially inactivated BoNT/A was determined. A suitable density of cells from a SiMa cell line was plated into the wells of poly-D-lysine coated 96-well cell culture plates containing 100 .mu.L of a serum-free medium containing Minimum Essential Medium, 2 mM GlutaMAX.TM. I with Earle's salts, 1.times.B27 supplement, 1.times.N2 supplement, 0.1 mM Non-Essential Amino Acids, 10 mM HEPES and 25 .mu.g/mL GT1b. Cells were incubated in a 37.degree. C. incubator under 5% carbon dioxide until the cells differentiated, as assessed by standard and routine morphological criteria, such as growth arrest and neurite extension (approximately 3 days). The media was aspirated from each well and replaced with fresh media containing either 1) 0 (untreated sample), 0.03 pM, 0.1 pM, 0.31 pM, 0.93 pM, 2.78 pM, 8.33 pM, and 25 pM, of a BoNT/A complex; 2) 0, 0.14 nM, 0.41 nM, 1.23 nM, 3.7 nM, 11.11 nM, 33.33 nM, and 100 nM of an inactive BoNT/A (iBoNT/A); or 3) 0, 0.14 nM, 0.41 nM, 1.23 nM, 3.7 nM, 11.11 nM, 33.33 nM, and 100 nM of an LH.sub.N/A fragment. The iBoNT/A contains a mutation in the zinc binding domain of the light chain that completely inactivates the metalloprotease activity of the neurotoxin, see, e.g., Liqing Zhou, et al., Expression and Purification of the Light Chain of Botulinum Neurotoxin A: A Single Mutation Abolishes its Cleavage of SNAP-25 and Neurotoxicity after Reconstitution with the Heavy Chain, Biochemistry 34: 15175-15181 (1995), which is hereby incorporated by reference in its entirety. The LH.sub.N/A fragment lacks the binding domain, but contains an intact translocation domain and light chain, see, e.g., Clifford C. Shone, et al., Recombinant Toxin Fragments, U.S. Pat. No. 6,461,617, which is hereby incorporated by reference in its entirety. After 24 hrs treatment, the cells were washed, incubated for an additional two days without toxin to allow for the cleavage of SNAP-25 substrate, and harvested as described above in Section 1. After harvesting, the protein concentrations of cell lysates, detection of SNAP-25 cleavage product by ECL sandwich ELISA performed, and the EC.sub.50 calculated as described above. The results indicate that the binding affinity of cells for iBoNT/A and LH.sub.N/A (EC.sub.50>100 nM) are at least 60,000 lower than the binding affinity for BoNT/A (EC.sub.50=1.6 pM) (FIG. 7). No SNAP-25 cleavage product was detected in cells treated with iBoNT/A at all concentrations tested. Although a low amount of SNAP-25 cleavage product was detected in cells treated with the highest dose of the LH.sub.N/A fragment, this activity is due to non-specific uptake of this fragment due to the activity of the translocation domain. Thus, the results indicate that the immuno-based methods of detecting BoNT/A activity disclosed in the present specification can measure all the steps involved in the intoxication process whereby a BoNT/A proteolytically cleaves a SNAP-25 substrate and encompasses the binding of a BoNT/A to a BoNT/A receptor, the internalization of the neurotoxin/receptor complex, the translocation of the BoNT/A light chain from an intracellular vesicle into the cytoplasm and the proteolytic cleavage of a SNAP-25.

Example VI

Immuno-Based Method of Detecting BoNT/a Activity Using ECL Sandwich ELISA

[0242] The following example illustrates immuno-based methods of detecting BoNT/A activity by detecting a SNAP-25 cleavage product using a .alpha.-SNAP-25 monoclonal antibody specific for a SNAP-25 cleavage product having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond using ECL sandwich ELISA.

[0243] To prepare a lysate from cells treated with a BoNT/A, a suitable density of cells from an established cell line was plated into the wells of 96-well tissue culture plates containing 100 .mu.L of a serum-free medium containing Minimum Essential Medium, 2 mM GlutaMAX.TM. I with Earle's salts, 1.times.B27 supplement, 1.times.N2 supplement, 0.1 mM Non-Essential Amino Acids, 10 mM HEPES and 25 .mu.g/mL GT1b (see Examples I and II). These cells were incubated in a 37.degree. C. incubator under 5% carbon dioxide until the cells differentiated, as assessed by standard and routine morphological criteria, such as growth arrest and neurite extension (approximately 3 days). The media from the differentiated cells was aspirated from each well and replaced with fresh media containing either 0 (untreated sample), 0.03 pM, 0.1 pM, 0.3 pM, 0.9 pM, 2.8 pM, 8.3 pM, and 25 pM of a BoNT/A complex. After a 24 hr treatment, the cells were washed, and incubated for an additional two days without toxin. To cells were harvested as described in Example V.

[0244] To prepare the .alpha.-SNAP-25 capture antibody solution, the .alpha.-SNAP-25 monoclonal antibody contained in the ascites from hybridoma cell line 2E2A6 was purified using a standard Protein A purification protocol To prepare the .alpha.-SNAP-25 detection antibody solution, .alpha.-SNAP-25 rabbit polyclonal antibody S9684 (Sigma, St. Louis, Mo.) was conjugated to Ruthenium(II)-tris-bipyridine-(4-methysulfonate) NHS ester labeling reagent (Meso Scale Discovery, Gaithersburg, Md.) according to the manufacturer's instructions (Meso Scale Discovery, Gaithersburg, Md.). The conjugation reaction, purification of labeled .alpha.-SNAP-25 antibody, concentration determination and storage were as described in Example V.

[0245] To prepare the solid phase support comprising the capture antibody that is specific for a SNAP-25 cleaved product, approximately 5 .mu.L of .alpha.-SNAP-25 monoclonal antibody 2E2A6 solution (20 .mu.g/mL in 1.times.PBS) was added to each well of a 96-well MSD High Bind plate and the solution is allowed to air dry in a biological safety cabinet for 2-3 hours in order to liquid evaporate the solution. The capture antibody-bound wells were then blocked and used directly to detect BoNT/A activity.

[0246] To detect the presence of a cleaved SNAP-25 product by ECL sandwich ELISA analysis, the Blocking Buffer from stored plates was aspirated from the wells, 25 .mu.L of a lysate from cells treated with BoNT/A was added to each well and the plates were incubated at 4.degree. C. for overnight. Plate wells were washed three times by aspirating the cell lysate and rinsing each well three times with 200 .mu.L 1.times.PBS, 0.1% TWEEN-20.RTM. (polyoxyethylene (20) sorbitan monolaureate). After washing, 25 .mu.l of 5 .mu.g/mL detection antibody solution comprising 2% Amersham Blocking Reagent in 1.times.PBS, 0.1% TWEEN-20.RTM. (polyoxyethylene (20) sorbitan monolaureate) was added to each well, the plate was sealed, and the sealed plate was incubated at room temperature at room temperature for 1 hour with shaking. After detection antibody incubation, the wells were washed three times with 200 .mu.L 1.times.PBS, 0.1% TWEEN-20.RTM. (polyoxyethylene (20) sorbitan monolaureate). After washing 150 .mu.L of 1.times.Read Buffer (Meso Scale Discovery, Gaithersburg, Md.) was added to each well and the plates were read using a SECTOR.TM. Imager 6000 Image Reader (Meso Scale Discovery, Gaithersburg, Md.). The collected data was analyzed and the EC.sub.50 calculated as described in Example V. A representative result is shown in FIG. 8. These results indicated that on average 1.0 pM of BoNT/A at the EC.sub.50 was detected (a range of about 0.3 pM to about 2.0 pM) with a signal-to-noise ratio for the lower asymptote of about 15:1 to about 20:1 and a signal-to-noise ratio for the upper asymptote of about 20:1 to about 500:1.

Example VII

Immuno-Based Method of Detecting BoNT/a Activity Using CL Sandwich ELISA

[0247] The following example illustrates immuno-based methods of detecting BoNT/A activity by detecting a SNAP-25 cleavage product using a .alpha.-SNAP-25 monoclonal antibody specific for a SNAP-25 having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond by CL sandwich ELISA.

[0248] Lysate from cells treated with a BoNT/A and the .alpha.-SNAP-25 capture antibody solution were prepared as described in Example VI.

[0249] To prepare the .alpha.-SNAP-25 detection antibody solution, .alpha.-SNAP-25 polyclonal antibody S9684 (Sigma, St. Louis, Mo.) was conjugated to Horseradish peroxidase (HRP) according to the manufacturer's instructions (Pierce Biotechnology, Inc., Rockford, Ill.). The conjugation reaction was performed by adding to 500 .mu.L of 1 mg/mL .alpha.-SNAP-25 polyclonal antibodies to a vial containing lyophilized activated peroxidase, mixing the components, and then adding 10 .mu.L of sodium cyanoborohydride. This reaction mixture was incubated at room temperature for 1 hour in a fume hood. After quenching the reaction, the labeled antibodies were purified using a standard spin column protocol and the protein concentration determined using a standard colorimetric protein assay. The absorbance of the .alpha.-SNAP-25 polyclonal antibody/HRP conjugate was measured at 455 nm using a spectrophotometer to determine the concentration in moles per liter. The .alpha.-SNAP-25 detection antibody solution was stored at 4.degree. C. until needed.

[0250] To prepare the solid phase support comprising the .alpha.-SNAP-25 capture antibody that is specific for the SNAP-25 cleaved product, approximately 100 .mu.L of .alpha.-SNAP-25 monoclonal antibody 2E2A6 solution (1 mg/mL in 1.times.PBS) was added to each well of a 96-well Greiner white plate and the plates were incubated at 4.degree. C. overnight, and then any excess antibody solution was discarded. The capture antibody-bound wells were then blocked by adding 150 .mu.l of Blocking Buffer comprising 2% Amersham Blocking Reagent (GE Life Sciences, Piscataway, N.J.) and 10% goat serum (VWR, West Chester, Pa.) at room temperature for 1 hour. The blocking buffer was discarded and the plates were blotted dry on paper towels by inverting and tapping. The capture antibody-bound wells were then blocked and used directly to detect BoNT/A activity.

[0251] To detect the presence of a cleaved SNAP-25 product by CL sandwich ELISA analysis, 50 .mu.L of a lysate from cells treated with BoNT/A was added to each well, the plate was sealed, and the sealed plate was incubated on a shaker rotating at 500 rpm at 4.degree. C. for 2-4 hours to overnight. Plate wells were washed three times by aspirating the cell lysate and rinsing each well three times with 200 .mu.l 1.times.PBS, 0.05% TWEEN-20.RTM. (polyoxyethylene (20) sorbitan monolaureate). After washing, 100 .mu.L of 1 mg/mL .alpha.-SNAP-25 polyclonal antibody/HRP detection antibody solution comprising 2% Amersham Blocking Reagent in 1.times.PBS, 0.1% TWEEN-20.RTM. (polyoxyethylene (20) sorbitan monolaureate) was added to each well, the plate was sealed, and the sealed plate was incubated on a shaker rotating at 650 rpm at room temperature for 1 hour. After detection antibody incubation, the wells were washed three times with 200 .mu.l 1.times.PBS, 0.05% TWEEN-20.RTM. (polyoxyethylene (20) sorbitan monolaureate). After washing 100 .mu.l of SuperSignal ELISA Pico 1:1 mixture (Pierce Biotechnology, Inc., Rockford, Ill.) was added to each well and the plates were read using a luminometer (Molecular Devices, Sunnyvale, Calif.) at 395 nm. The collected data was analyzed and the EC.sub.50 calculated as described in Example V. These results indicated that on average 1.0 pM of BoNT/A at the EC.sub.50 was detected (a range of about 0.3 pM to about 2.0 pM) with a signal-to-noise ratio for the lower asymptote of about 15:1 to about 20:1 and a signal-to-noise ratio for the upper asymptote of about 20:1 to about 500:1.

Example VIII

Immuno-Based Method of Detecting BoNT/a Activity Using Multiplex ECL Sandwich ELISA

[0252] The following example illustrates multiplex immuno-based methods of detecting BoNT/A activity by detecting a SNAP-25 cleavage product using a .alpha.-SNAP-25 monoclonal antibody specific for a SNAP-25 cleavage product and a second antibody pair for a different protein.

1. Preparation and Identification of Capture Antibody-Detection Antibody Pair for a Second Protein.

[0253] To obtain an untreated cell lysate for analysis, a suitable density of cells from a stock culture of SiMa cells were seeded into a T175 flask containing 40 mL of a growth medium containing 1.times.RPMI 1640, 10% FBS, 0.1 mM Non-Essential Amino Acids, 10 mM HEPES, 1 mM sodium pyruvate, and 100 U/100 .mu.g of pennicillin-streptomycin. These cells were incubated in a 37.degree. C. incubator under 5% carbon dioxide until the cells were approximately 70-90% confluent. The cells were washed and harvested by lysing in freshly prepared Triton X-100 Lysis Buffer (20 mM Tris pH 7.5, 150 mM sodium chloride, 0.001M EDTA, 1 mM EGTA, and 1% Triton-X-100) at 4.degree. C. for approximately 30 minutes with constant agitation. Lysed cells were centrifuged at approximately 3300-3330.times.g for 40 minutes at 8.degree. C. to eliminate debris using a bench-top centrifuge.

[0254] To identify an appropriate capture antibody-detection antibody pair for a second protein, an ECL sandwich ELISA analysis was conducted on 21 different combinations of capture and detection antibody pairs comprising of five different proteins (Table 13). The antibodies used were .alpha.-Syntaxin 1A-HPC mouse monoclonal antibody S0664 (Sigma, St. Louis, Mo.), .alpha.-GAPDH mouse monoclonal antibody MAB374 (Chemicon, Temecula, Calif.), .alpha.-Syntaxin 1 rabbit polyclonal antibody S1172-1 (Sigma, St. Louis, Mo.), .alpha.-GAPDH rabbit polyclonal antibody 2275-PC-1 (R & D Systems, Minneapolis, Minn.), .alpha.-Syntaxin 2 rabbit polyclonal antibody S5687 (Sigma, St. Louis, Mo.), .alpha.-human syntaxin 2 mouse monoclonal antibody MAB2936 (R & D Systems, Minneapolis, Minn.), .alpha.-mouse syntaxin 2 goat polyclonal antibody AF2568 (Sigma, St. Louis, Mo.), .alpha.-Syntaxin 2 rabbit polyclonal antibody AB5596 (Sigma, St. Louis, Mo.), .alpha.-Syntaxin 1 rabbit polyclonal antibody S1172-2 (Sigma, St. Louis, Mo.), a-h, m, r actin sheep polyclonal antibody AF4000 (R & D Systems, Minneapolis, Minn.), .alpha.-beta actin mouse monoclonal antibody A1978 (Sigma, St. Louis, Mo.), .alpha.-beta mouse polyclonal antibody actin A2228 (Sigma, St. Louis, Mo.), mouse .alpha.-GAPDH mouse monoclonal antibody G8795 (Sigma, St. Louis, Mo.), .alpha.-GAPDH rabbit polyclonal antibody G9595 (Sigma, St. Louis, Mo.).

[0255] To prepare the second protein capture antibody solution, the monoclonal antibodies were purchased as purified antibodies. To prepare the second protein detection antibody solution, the appropriate antibody was conjugated to Ruthenium(II)-tris-bipyridine-(4-methysulfonate) NHS ester labeling reagent (Meso Scale Discovery, Gaithersburg, Md.) according to the manufacturer's instructions (Meso Scale Discovery, Gaithersburg, Md.). The conjugation reaction was performed by adding 30 .mu.L of distilled water reconstituted MSD SULFO-TAG.TM. stock solution to 200 .mu.L of 2 mg/mL polyclonal antibodies and incubating the reaction at room temperature for 2 hours in the dark. The labeled antibodies were purified using a standard spin column protocol and the protein concentration determined using a standard colorimetric protein assay. The absorbance of the antibody/MSD SULFO-TAG.TM. conjugate was measured at 455 nm using a spectrophotometer to determine the concentration in moles per liter. The detection antibody solution was stored at 4.degree. C. until needed.

[0256] To prepare the solid phase support comprising the capture antibody that is specific for a SNAP-25 cleaved product, approximately 5 .mu.L of .alpha.-SNAP-25 monoclonal antibody 2E2A6 solution (20 .mu.g/mL in 1.times.PBS) was added to each well of a 96-well MSD High Bind plate and the solution is allowed to air dry in a biological safety cabinet for 2-3 hours in order to liquid evaporate the solution, and then the plates were sealed and stored at 4.degree. C. until needed. The dried capture antibody-bound wells were then blocked by adding 150 .mu.L of Blocking Buffer comprising of 3% BSA (Pierce, Rockford, Ill.) 10% goat serum (Rockland Immunochemicals, Gilbertsville, Pa.), and Difco 1% skim milk (BD BioSciences Franklin Lakes, N.J.) in 0.05% Tween-20 PBS at room temperature for 1-2 hours.

[0257] To detect the presence of protein by ECL sandwich ELISA analysis, the Blocking Buffer from stored plates was aspirated from the wells, 25 .mu.L of a lysate from cells treated with BoNT/A, as described above, was added to each well and the plates were incubated at 4.degree. C. for overnight. Plate wells were washed three times by aspirating the cell lysate and rinsing each well three times with 200 .mu.L 1.times.PBS, 0.1% TWEEN-20.RTM. (polyoxyethylene (20) sorbitan monolaureate). After washing, 25 .mu.L of 5 .mu.g/mL the appropriate second protein detection antibody solution, resuspended in the blocking buffer as described above, was added to each well, the plate was sealed, and the sealed plate was incubated at room temperature for about 1 hour with shaking. After detection antibody incubation, the wells were washed three times with 250 .mu.L 1.times.PBS, 0.1% TWEEN-20.RTM. (polyoxyethylene (20) sorbitan monolaureate). After washing 150 .mu.L of 1.times. Read Buffer (Meso Scale Discovery, Gaithersburg, Md.) was added to each well and the plates were read using a SECTOR.TM. Imager 6000 Image Reader (Meso Scale Discovery, Gaithersburg, Md.). A ratio was calculated by dividing the signal obtained from the untreated cell lysates for each antibody-pair by the signal obtained for the lysis buffer control (0 nM dose) for each antibody-pair (Table 13). These results indicated that among the twenty-one different combinations of antibody pairs tested, only two antibody pairs had signal-to-noise ratios above 10:1 for the higher dose tested: Pair No. 16 .alpha.-GAPDH mouse monoclonal antibody MAB374 and .alpha.-GAPDH rabbit polyclonal antibody RDS2275-PC-1; and Pair 21: .alpha.-GAPDH mouse monoclonal antibody MAB374 and .alpha.-GAPDH rabbit polyclonal antibody G9545. The .alpha.-GAPDH mouse monoclonal antibody MAB374 and .alpha.-GAPDH rabbit polyclonal antibody G9545 pair was selected as the second protein capture antibody-detection antibody pair for the multiplex ECL sandwich ELISA.

TABLE-US-00012 TABLE 13 Screening of Second Protein Antibody Combinations Signal/Noise Antibody Detection Ratio Pair No. Capture Antibody Detection Antibody of Protein (lysate/buffer) 1 .alpha.-syntaxin 2 S5687 pAb .alpha.-syntaxin 2 MAB2936 No 0.92 mAb 2 .alpha.-syntaxin 2 AF2568 pAb .alpha.-syntaxin 2 AB5596 pAb No 1.1 3 .alpha.-syntaxin 2 AF2568 .alpha.-syntaxin 2 S5687 pAb No 1.11 4 .alpha.-syntaxin 2 AF2936 pAb .alpha.-syntaxin 2 AB5596 pAb Yes 1.63 5 .alpha.-syntaxin 2 AF2936 pAb .alpha.-syntaxin 2 S5687 pAb Yes 1.6 6 .alpha.-syntaxin 2 AB5596 pAb .alpha.-syntaxin 2 S5687 pAb No 0.82 7 .alpha.-syntaxin 2 AB5596 pAb .alpha.-syntaxin 2 MAB2936 No 0.87 mAb 8 .alpha.-syntaxin 2 MAB2936 .alpha.-syntaxin 2 AB5596 pAb Yes 1.2 mAb 9 .alpha.-syntaxin 2 MAB2936 .alpha.-syntaxin 2 S5687 pAb No 1.07 mAb 10 .alpha.-syntaxin S0664 mAb .alpha.-syntaxin 1 S1172-1 pAb Yes 4.23 11 .alpha.-syntaxin S0664 mAb .alpha.-syntaxin 1 S1172-2 pAb No 1.21 12 .alpha.-syntaxin 1 S1172-1 pAb .alpha.-syntaxin S0664 mAb Yes 5.5 13 .alpha.-syntaxin 1 S1172-2 pAb .alpha.-syntaxin S0664 mAb Yes 2.5 14 .alpha.-h,m,r actin AF4000 pAb .alpha.-beta actin A 1978 mAb No 1.04 15 .alpha.-h,m,r actin AF4000 pAb .alpha.-beta actin A2228 mAb No 1.08 16 .alpha.-GAPDH MAB374 mAb .alpha.-GAPDH 2275-PC-1 pAb Yes 20.04 17 .alpha.-GAPDH MAB374 mAb .alpha.-GAPDH G8795 mAb No 0.89 18 .alpha.-GAPDH 2275-PC-1 pAb .alpha.-GAPDH MAB374 mAb No 1.08 19 .alpha.-GAPDH 2275-PC-1 pAb .alpha.-GAPDH G8795 mAb Yes 1.27 20 .alpha.-GAPDH G8795 mAb .alpha.-GAPDH 2275-PC-1 pAb Yes 2.74 21 .alpha.-GAPDH MAB374 mAb .alpha.-GAPDH G9545 pAb Yes .gtoreq.100

2. Immuno-Based Method of Detecting BoNT/a Activity Using Multiplex ECL Sandwich ELISA.

[0258] To obtain a BoNT/A treated cell lysate for analysis, a suitable density of cells from a stock culture of a SiMa cell line were seeded into a poly-D-lysine 96-well plate containing a serum-free medium containing Minimum Essential Medium, 2 mM GlutaMAX.TM. I with Earle's salts, 1.times.B27 supplement, 1.times.N2 supplement, 0.1 mM Non-Essential Amino Acids, 10 mM HEPES. These cells were incubated in a 37.degree. C. incubator under 5% carbon dioxide until the cells differentiated, as assessed by standard and routine morphological criteria, such as growth arrest and neurite extension (approximately 3 days). The media was aspirated from each well and replaced with fresh media containing either 0 (untreated sample), 0.67 U/mL, 2.35 U/mL, 8.23 U/mL, 28.82 U/mL, 101 U/mL, 353 U/mL of a BoNT/A complex. After a 24 hr treatment, the cells were washed, incubated for an additional two days without toxin. The cells were washed, harvested, and processed as described above in Section 1.

[0259] The .alpha.-SNAP-25 capture antibody solution and the .alpha.-SNAP-25 detection antibody solution, were prepared as described in Example VII. To prepare the .alpha.-GAPDH capture antibody solution, the .alpha.-GAPDH monoclonal antibody mouse MAB374 (Chemicon, Temecula, Calif.) was prepared as described in Section 1 above. To prepare the .alpha.-GAPDH detection antibody solution, .alpha.-GAPDH rabbit polyclonal antibody G9545 (Sigma, St. Louis, Mo.) was conjugated to Ruthenium(II)-tris-bipyridine-(4-methysulfonate) NHS ester labeling reagent (Meso Scale Discovery, Gaithersburg, Md.) according to the manufacturer's instructions (Meso Scale Discovery, Gaithersburg, Md.). The conjugation reaction, purification of labeled .alpha.-SNAP-25 antibody, concentration determination and storage were as described in Section 1 above.

[0260] To prepare the solid phase support comprising the .alpha.-SNAP-25 capture antibody and the .alpha.-GAPDH capture antibody, approximately 2.5 nL of the .alpha.-SNAP-25 capture antibody solution (45 .mu.g/mL in 1.times.PBS) and 2.5 nL of the .alpha.-GAPDH capture antibody solution (120 pg/mL in 1.times.PBS) were added to each well of a 96-well MSD High Bind plate in a muliplex format using a robotic system. The solution is allowed to air dry in a biological safety cabinet for at least 2-3 hours in order to liquid evaporate the solution. The capture antibody-bound wells were then blocked and used directly to detect BoNT/A activity and the GAPDH protein.

[0261] To detect the presence of SNAP-25 cleavage product by multiplex ECL sandwich ELISA analysis, the Blocking Buffer from stored plates was aspirated from the wells, 25 .mu.L of a lysate from cells treated with BoNT/A, as described above, was added to each well and the plates were incubated at 4.degree. C. for overnight. Plate wells were washed three times by aspirating the cell lysate and rinsing each well three times with 200 .mu.L 1.times.PBS, 0.1% TWEEN-20.RTM. (polyoxyethylene (20) sorbitan monolaureate). After washing, 25 .mu.L of 5 .mu.g/mL the .alpha.-SNAP-25 detection antibody solution and 25 .mu.L of 5 .mu.g/mL the .alpha.-GAPDH detection antibody solution, as described above, was added to each well, the plate was sealed, and the sealed plate was incubated at room temperature for about 1 hour with shaking. After detection antibody incubation, the wells were washed three times with 250 .mu.L 1.times.PBS, 0.1% TWEEN-20.RTM. (polyoxyethylene (20) sorbitan monolaureate). After washing 150 .mu.L of 1.times.Read Buffer (Meso Scale Discovery, Gaithersburg, Md.) was added to each well and the plates were read using a SECTOR.TM. Imager 6000 Image Reader (Meso Scale Discovery, Gaithersburg, Md.). The collected data was analyzed and the relative potency from the normalized data is calculated as described in Example V, except that PLA 2.0 software (Stegmann Systems, GmbH, Germany) was used.

[0262] As a comparison, the detection of SNAP-25 cleavage product was also performed using the singleplex ECL sandwich ELISA as described in Example VI.

[0263] The results indicated that the SNAP-25 data obtained from the singleplex ECL sandwich ELISA, or from the non-normalized SNAP-25 data obtained from the multiplex ECL sandwich ELISA, revealed one outlier sample dose that did not fit into the dose-response curve. However, normalization of the SNAP-25 data against the GAPDH data gave a better curve fit and the potency was closer to the expected value.

Example IX

Immuno-Based Method of Detecting BoNT/a Activity Using Multiplex EC Sandwich ELISA

[0264] The following example illustrates multiplex immuno-based methods of detecting BoNT/A activity by detecting a SNAP-25 cleavage product using a .alpha.-SNAP-25 monoclonal antibody specific for a SNAP-25 cleavage product and a second antibody pair for a different protein.

[0265] The lysate from cells treated with a BoNT/A was prepared as described in Example VI. The .alpha.-SNAP-25 capture antibody solution, the .alpha.-SNAP-25 detection antibody solution, and the .alpha.-SNAP-25 solid phase support were prepared as described in Example VII.

[0266] To prepare .alpha.-GAPDH capture antibody solution, .alpha.-GAPDH monoclonal antibody MAB374 (Millipore, Billerica, Mass.) was purchased as a purified antibody. To prepare the .alpha.-GAPDH detection antibody solution, an .alpha.-GAPDH polyclonal antibody G9545 (Sigma, St. Louis, Mo.) was conjugated to Horseradish peroxidase (HRP) according to the manufacturer's instructions (Pierce Biotechnology, Inc., Rockford, Ill.). The conjugation reaction, concentration determination and storage were as described in Example VII.

[0267] To prepare the solid phase support comprising a second capture antibody specific for the second protein, approximately 100 .mu.L of monoclonal antibody solution comprising 1 .mu.g/mL .alpha.-GAPDH monoclonal antibody MAB374 was added to each well of a 96-well Greiner white plate and the plates were incubated at 4.degree. C. overnight, and then any excess antibody solution was discarded. The .alpha.-GAPDH capture antibody-bound wells were then blocked by adding 150 .mu.l of Blocking Buffer comprising 2% Amersham Blocking Reagent (GE Life Sciences, Piscataway, N.J.) and 10% goat serum (VWR, West Chester, Pa.) at room temperature for 1 hour. The blocking buffer was discarded and the plates were blotted dry on paper towels by inverting and tapping. The capture antibody-bound wells were then blocked and used directly to detect BoNT/A activity.

[0268] To detect the presence of a cleaved SNAP-25 product by multiplex CL sandwich ELISA analysis, 50 .mu.L of cell lysates from cells treated with BoNT/A was added to each well of the .alpha.-SNAP-25 capture antibody solid phase support and the .alpha.-GAPDH capture antibody solid phase support, the plate was sealed, and the sealed plate was incubated on a shaker rotating at 500 rpm at 4.degree. C. for 2-4 hours to overnight. Plate wells were washed three times by aspirating the cell lysate and rinsing each well three times with 200 .mu.l 1.times.PBS, 0.05% TWEEN-20.RTM. (polyoxyethylene (20) sorbitan monolaureate). After washing, 100 .mu.L of a detection antibody solution comprising 2% Amersham Blocking Reagent in 1.times.PBS, 0.1% TWEEN-20.RTM. (polyoxyethylene (20) sorbitan monolaureate), and 1 mg/mL .alpha.-SNAP-25 polyclonal antibody/HRP was added to each well of the .alpha.-SNAP-25 capture antibody solid phase support, the plate was sealed, and the sealed plate was incubated on a shaker rotating at 650 rpm at room temperature for 1 hour. Similarly, 100 .mu.L of a detection antibody solution comprising 2% Amersham Blocking Reagent in 1.times.PBS, 0.1% TWEEN-20.RTM. (polyoxyethylene (20) sorbitan monolaureate), and 0.25 mg/mL .alpha.-GAPDH G9545 polyclonal antibody/HRP (Sigma Co., St Louis, Mo.) was added to each well of the .alpha.-GAPDH capture antibody solid phase support, the plate was sealed, and the sealed plate was placed on a shaker rotating at 650 rpm at room temperature for 1 hour. After detection antibody incubation, the wells were washed three times with 200 .mu.l 1.times.PBS, 0.05% TWEEN-20.RTM. (polyoxyethylene (20) sorbitan monolaureate). After washing 100 .mu.l of SuperSignal ELISA Pico 1:1 mixture (Pierce Biotechnology, Inc., Rockford, Ill.) was added to each well and the plates were read using a luminometer (Molecular Devices, Sunnyvale, Calif.) at 395 nm. The collected data was analyzed and the EC.sub.50 calculated as described in Example V. The results indicated that the data points collected for the amounts of .alpha.-SNAP-25 antibody-antigen complex detected were a better fit after normalization to the amounts of .alpha.-GAPDH antibody-antigen complex detected, thereby producing a more accurate reading. These results indicated that on average 1.0 pM of BoNT/A at the EC.sub.50 was detected (a range of about 0.3 pM to about 2.0 pM) with a signal-to-noise ratio for the lower asymptote of about 15:1 to about 20:1 and a signal-to-noise ratio for the upper asymptote of about 20:1 to about 500:1.

[0269] A similar design can be used for multiplex immuno-based methods of detecting BoNT/A activity by detecting a SNAP-25 cleavage product using a .alpha.-SNAP-25 monoclonal antibody specific for a SNAP-25 cleavage product having a carboxyl-terminus at the P.sub.1 residue of the BoNT/A cleavage site scissile bond using ECL sandwich ELISA with the same .alpha.-GAPDH antibody pair.

Example X

Immuno-Based Method to Detect Picomolar Amounts of BoNT/A

[0270] The following example illustrates how to perform immuno-based methods of detecting BoNT/A activity that can detect picomolar amounts of the BoNT/A pharmaceutical product, such as, e.g., BOTOX.RTM. DYSPORT.RTM./RELOXIN.RTM., PURTOX.RTM., XEOMIN.RTM., NEURONOX.RTM., or BTX-A.

1. Immuno-Based Method of Detecting BoNT/a Using ECL Sandwich ELISA.

[0271] To prepare a lysate from cells treated with a BoNT/A, approximately 50,000 to 150,000 cells from an established cell line were plated into the wells of 96-well tissue culture poly-D-lysine plates containing 100 .mu.L of a serum-free medium containing Minimum Essential Medium, 2 mM GlutaMAX.TM. I with Earle's salts, 1.times.B27 supplement, 1.times.N2 supplement, 0.1 mM Non-Essential Amino Acids, 10 mM HEPES and 25 .mu.g/mL GT1b (see Examples I and II). These cells were incubated in a 37.degree. C. incubator under 5% carbon dioxide until the cells differentiated, as assessed by standard and routine morphological criteria, such as growth arrest and neurite extension (approximately 2 to 3 days). The media from the differentiated cells was aspirated from each well and replaced with fresh media containing either 0 (untreated sample), 0.03 pM, 0.1 pM, 0.3 pM, 0.9 pM, 2.8 pM, 8.3 pM, or 25 pM of a BoNT/A pharmaceutical product reconstituted in a sodium chloride free solution; or 0 (untreated sample), 0.7 U/mL, 2.1 U/mL, 6.2 U/mL, 18.5 U/mL, 55.6 U/mL, 166.7 U/mL or 500 U/mL of a BoNT/A pharmaceutical product reconstituted in a sodium chloride free medium. Because the BoNT/A pharmaceutical product contains sodium chloride, its addition to the culture medium resulted in a hypertonic media that was detrimental to cell viability. To circumvent the hypertonicity issue, 200 .mu.L of MEM media made without sodium chloride was used to reconstitute the BoNT/A pharmaceutical product giving a final concentration of 25 pM BoNT/A (500 U/mL). The matrix was kept constant for all concentrations along the dose-response curve by adding sodium chloride in the media used to make the dilutions match the amount of excipients present at the highest concentration used (25 pM or 500 U/mL). After a 24 hr treatment, the cells were washed, and incubated for an additional two days without toxin. To harvest the cells, the medium was aspirated, washed with 1.times.PBS, and lysed by adding 30 .mu.l of Lysis Buffer comprising 50 mM HEPES, 150 mM NaCl, 1.5 mM MgCl.sub.2, 1 mM EGTA, 1% Triton X-100 to each well, and the plate incubated on a shaker rotating at 500 rpm for 30 minutes at 4.degree. C. The plate was centrifuged at 4000 rpm for 20 minutes at 4.degree. C. to pellet cellular debris and the supernatant was transferred to a capture antibody coated 96-well plate to perform the detection step.

[0272] The .alpha.-SNAP-25 capture antibody solution, the .alpha.-SNAP-25 detection antibody solution, and the solid phase support comprising the capture antibody that is specific for a SNAP-25 cleaved product were prepared as described in Example VI.

[0273] To detect the presence of a cleaved SNAP-25 product by ECL sandwich ELISA analysis, the Blocking Buffer from stored plates was aspirated, 25 .mu.L of a lysate from cells treated with BoNT/A was added to each well and the plates were incubated at 4.degree. C. for either 2 hrs or 24 hrs. Plate wells were washed three times by aspirating the cell lysate and rinsing each well three times with 200 .mu.L 1.times.PBS, 0.1% TWEEN-20.RTM. (polyoxyethylene (20) sorbitan monolaureate). After washing, 25 .mu.l of 5 .mu.g/mL .alpha.-SNAP-25 detection antibody solution comprising 2% Amersham Blocking Reagent in 1.times.PBS, 0.1% TWEEN-20.RTM. (polyoxyethylene (20) sorbitan monolaureate) was added to each well, the plate was sealed, and the sealed plate was incubated at room temperature for 1 hour with shaking. After .alpha.-SNAP-25 detection antibody incubation, the wells were washed three times with 200 .mu.L 1.times.PBS, 0.1% TWEEN-20.RTM. (polyoxyethylene (20) sorbitan monolaureate). After washing, the plates were processed, collected data was analyzed, and the EO.sub.50 calculated as described in Example V. These results indicated that on average 1.0 pM of BoNT/A at the EC.sub.50 was detected (a range of about 0.3 pM to about 2.0 pM) with a signal-to-noise ratio for the lower asymptote of about 15:1 to about 20:1 and a signal-to-noise ratio for the upper asymptote of about 20:1 to about 500:1 (FIG. 9). This method can also be performed in a multiplex fashion as described in Example VIII.

2. Immuno-Based Method of Detecting BoNT/a Using CL Sandwich ELISA.

[0274] Lysate from cells treated with a BoNT/A and the .alpha.-SNAP-25 capture antibody solution will be prepared as described in Example VI. The .alpha.-SNAP-25 detection antibody solution and solid phase support comprising the capture antibody that is specific for a SNAP-25 cleaved product will be prepared as described in Example VII.

[0275] To detect the presence of a cleaved SNAP-25 product by CL sandwich ELISA analysis, 25 .mu.L of a lysate from cells treated with BoNT/A will be added to each well, the plate was sealed, and the sealed plate was incubated on a shaker rotating at 500 rpm at 4.degree. C. for either 2 hrs or 24 hrs. Plate wells will be washed three times by aspirating the cell lysate and rinsing each well three times with 200 .mu.l 1.times.PBS, 0.05% TWEEN-20.RTM. (polyoxyethylene (20) sorbitan monolaureate). After washing, 100 .mu.L of 1 mg/mL .alpha.-SNAP-25 polyclonal antibody/HRP detection antibody solution comprising 2% Amersham Blocking Reagent in 1.times.PBS, 0.1% TWEEN-20.RTM. (polyoxyethylene (20) sorbitan monolaureate) will be added to each well, the plate was sealed, and the sealed plate was incubated on a shaker rotating at 650 rpm at room temperature for 1 hour. After detection antibody incubation, the wells will be washed three times with 200 .mu.l 1.times.PBS, 0.05% TWEEN-20.RTM. (polyoxyethylene (20) sorbitan monolaureate). After washing 100 .mu.l of SuperSignal ELISA Pico 1:1 mixture (Pierce Biotechnology, Inc., Rockford, Ill.) will be added to each well and the plates will be read using a luminometer (Molecular Devices, Sunnyvale, Calif.) at 395 nm. The collected data will be analyzed and the EC.sub.50 will be calculated as described in Example V. This method can also be performed in a multiplex fashion as described in Example VIII.

Example XI

Immuno-Based Method to Detect Neutralizing .alpha.-BoNT/a Antibodies

[0276] The following example illustrates how to perform an immuno-based method that can detect the presence of neutralizing .alpha.-BoNT/A antibodies.

[0277] BoNT/A, is currently used for a wide range of medical indications including muscle hyperactivity, ophthalmologic, gastrointestinal, urologic, and cosmetic. With repeated long-term treatment of BoNT/A, a patient may develop neutralizing .alpha.-BoNT/A antibodies to the toxin leading to immunoresistance. Neutralizing .alpha.-BoNT/A antibodies inhibit BoNT/A activity by stopping the toxin's uptake into neuronal cells by binding to the binding domain (H.sub.C) and/or the translocation domain (H.sub.N) of BoNT/A. Some studies have suggested that up to 5-10% of patients repeatedly treated for dystonia with formulations of BoNT/A have immunoresistance due to the development of neutralizing .alpha.-BoNT/A antibodies. The established assay to determine the presence of the neutralizing .alpha.-BoNT/A antibodies in patient's blood is the mouse protection assay (MPA). Currently, BoNT/A is incubated with a patient's serum prior to injection into mice. The presence of antibodies is manifested by a decreased response to the neurotoxin in the animal. Since the MPA is an in vivo based assay, it would be more cost and time efficient if it was replaced with a cell-based assay.

[0278] To detect the presence or absence of neutralizing .alpha.-BoNT/A antibodies, the immuno-based methods of determining BoNT/A activity disclosed in the present specification can be used. One way is to determine the amount of SNAP-25 cleavage product present after treatment with various concentrations of BoNT/A using a Western blot detection method, the other way was to use an ECL sandwich ELISA detection method.

[0279] To prepare a sample comprising neutralizing .alpha.-BoNT/A antibodies and a negative control sample known to lack .alpha.-BoNT/A neutralizing antibodies, serum was isolated from blood of different individuals. Individuals declining immunizations were referred to as naive individuals. Individuals accepting immunization were referred to as immunized individuals. The blood was drawn into a serum tube with a clot activator (BD Biosciences, Bedford, Mass.). Serum was obtained by centrifugation of the blood at 1000.times.g for 10 minutes at 4.degree. C. The serum of two donors was obtained: one individual was immunized to BoNT/A while the other was not.

[0280] To prepare a lysate from cells treated with a sample comprising BoNT/A, SiMa cells were seeded in a poly-D-lysine 96-well plate and differentiated as described in Example VI. The human serums were serially diluted 1:100-1:152,000 by 2.5 fold increments using serum-free media. The BoNT/A was suspended in 0.5 mL SiMa culture media at a concentration of 10 pM. The media containing BoNT/A and .alpha.-BoNT/A antibodies were mixed and incubated for 15 min or 1 hr at room temperature. The cells were treated with BoNT/A with human serum for 2 hr followed by a 15 hr incubation in fresh growth media. The cells were also treated for 15 hr with no additional incubation time.

[0281] To detect the presence of a cleaved SNAP-25 product by Western blot analysis, the media was aspirated from each well, the cells suspended in 50 .mu.L of SDS-PAGE loading buffer, and then heated to 95.degree. C. for 5 minutes. An aliquot from each harvested sample was analyzed by Western blot as described in Example I, except that harvested samples are separated by SDS-PAGE using 12% 26-well Criterion gels (Bio-Rad Laboratories, Hercules, Calif.), and the rabbit polyclonal .alpha.-SNAP-25.sub.197 antibody serum was used as the primary antibody (see Example IV). The results indicate that test samples resulted in reduced cleavage of SNAP25 when compared to the negative control sample, demonstrating that the serum from the immunized individual contained neutralizing .alpha.-BoNT/A antibodies.

[0282] To detect the presence of a cleaved SNAP-25 product by ECL Sandwich ELISA, the media was removed from each well and the cells were lysed as described in Example V. The .alpha.-SNAP-25 capture antibody solution, the .alpha.-SNAP-25 detection antibody solution, and the .alpha.-SNAP-25 solid phase support were prepared as described in Example VII. Supernatants were transferred to the .alpha.-SNAP-25 solid phase support and an ECL sandwich ELISA assay was performed as detailed in Example V. The collected data was analyzed and the EC.sub.59 calculated as described in Example V, except that the EC.sub.50 is the serum dilution needed to inhibit the activity of the BoNT/A to 1/2 its maximum and the ratio of maximal signal (Signal.sub.Max) to minimum signal (Signal.sub.Min) was obtained by dividing the SNAP-25 cleavage product signal obtained with the highest dilution of serum by the signal obtained with the lowest serum dilution.

[0283] The results indicate that the presence of neutralizing .alpha.-BoNT/A in human serum could be detected. The activity of the BoNT/A complex incubated in serum from the immunized individual decreased as the serum dilution decreased, whereas, the presence of naive serum had no impact on the assay at every dilution tested. This assay can be performed using a formulated BoNT/A pharmaceutical product, a bulk BoNT/A complex, or a purified neurotoxin.

Example XII

Immuno-Based Method to Detect BoNT/a Activity Using Engineered Cells

[0284] The following example illustrates how to introduce a polynucleotide molecule encoding a BoNT/A receptor into cells from an established cell line to further improve susceptibility to BoNT/A intoxication or improve BoNT/A uptake capacity.

[0285] To introduce an exogenous BoNT/A receptor into cells comprising an established cell line, an expression construct comprising a polynucleotide molecule of SEQ ID NO: 130 encoding the FGFR2 of SEQ ID NO: 59, or a polynucleotide molecule of SEQ ID NO: 139 encoding the FGFR3 of SEQ ID NO: 25, was transfected into cells from an established cell line by a cationic lipid method. A suitable density (about 5.times.10.sup.6 cells) of cells from an established cell line are plated in a 100 mm tissue culture dish containing 5 mL of complete culture media and grown in a 37.degree. C. incubator under 5% carbon dioxide until the cells reached a density appropriate for transfection. A 3 mL transfection solution is prepared by adding 1.5 mL of OPTI-MEM Reduced Serum Medium containing 60 .mu.L of LipofectAmine 2000 (Invitrogen, Carlsbad, Calif.) incubated at room temperature for 5 minutes to 1.5 mL of OPTI-MEM Reduced Serum Medium containing 24 pg of an expression construct encoding a FGFR2 or a FGFR3, or a control expression construct encoding a green fluorescent protein (GFP). This transfection mixture was incubated at room temperature for approximately 30 minutes. The complete media is replaced with the 3 mL transfection solution and the cells are incubated in a 37.degree. C. incubator under 5% carbon dioxide for approximately 8 hours. Transfection media is replaced with 3 mL of fresh complete culture media and the cells are incubated in a 37.degree. C. incubator under 5% carbon dioxide for approximately 24 hours. Media is replaced with 3 mL of fresh complete culture media containing approximately 1 mM G418 (Invitrogen, Carlsbad, Calif.). Cells are incubated in a 37.degree. C. incubator under 5% carbon dioxide for approximately 1 week, the old media is replaced with fresh complete culture media containing 0.5 mM G418. Once antibiotic-resistant colonies are established, resistant clones are replated to new 100 mm culture plates containing fresh complete culture media, supplemented with approximately 0.5 mM G418 until these cells reached a density of 6 to 20.times.10.sup.5 cells/mL.

[0286] To determine if the overexpression of BoNT/A receptors improved cell susceptibility to BoNT/A intoxication or improved BoNT/A uptake capacity, a dose-response curve was generated using cells treated with different doses of a BoNT/A complex. To prepare a lysate from cells treated with a BoNT/A, a suitable density of cells from an established transfected cell line was plated into the wells of 96-well tissue culture plates containing 100 .mu.L of an appropriate serum-free medium (Table 5). These cells were incubated in a 37.degree. C. incubator under 5% carbon dioxide until the cells differentiated, as assessed by standard and routine morphological criteria, such as growth arrest and neurite extension (approximately 3 days). The media from the differentiated cells was aspirated from each well and replaced with fresh media containing either 0 (untreated sample), 0.01 nM, 0.04 nM, 0.12 nM, 0.37 nM, 1.1 nM, 3.3 nM, and 10 nM of a BoNT/A complex for cells comprising a SiMa or a PC12 transfected cell line; and 0 (untreated sample), 0.14 nM, 0.40 nM, 1.2 nM, 3.7 nM, 11 nM, 33 nM, and 100 nM of a BoNT/A complex for cells comprising a Neuro-2a transfected cell line. The cells were treated with BoNT/A containing media for 6 hrs followed by incubation with fresh media for 15 hrs and harvested by adding 40 .mu.L of 2.times.SDS-PAGE loading buffer and heating the plate to 95.degree. C. for 5 min.

[0287] To detect for the presence of SNAP-25 cleavage product, an aliquot from each harvested sample was analyzed by Western blot as described in Example I, except that harvested samples are separated by SDS-PAGE using 12% 26-well Criterion gels (Bio-Rad Laboratories, Hercules, Calif.), and the following primary antibodies were used a 1:1,000 dilution of rabbit polyclonal .alpha.-SNAP-25 antibody serum (Example IV) (AGN, polyclonal antibody), a 1:500 dilution of .alpha.-FGFR2 rabbit polyclonal C-17 (Santa Cruz Biotechnology, Santa Cruz, Calif.), or a 1:500 dilution of .alpha.-FGFR3 rabbit polyclonal C-15 (Santa Cruz Biotechnology, Santa Cruz, Calif.). The intensity of the protein of interest from each sample was calculated using Image Quant (GE Healthcare, Piscataway, N.J.) and the EC.sub.50 for each of the cells lines was estimated using SigmaPlot software.

[0288] The results indicate that cells transfected with FGFR2 or FGFR3 were more sensitive to BoNT/A than cells transfected with GFP and also showed a higher level of SNAP-25 cleavage (Table 14). The EC.sub.50 values for cells over-expressing FGFR2 or FGFR3 were lower than the EC.sub.50 values exhibited by cells over-expressing GFP, indicating that introduction of FGFR2 or FGFR3 improved cell susceptibility to BoNT/A intoxication or improved BoNT/A uptake capacity.

TABLE-US-00013 TABLE 14 Effects of Introducing Exogenous BoNT/A Receptors on Cell Susceptibilty to BoNT/A Intoxication or BoNT/A Uptake Cells Transfected Gene EC.sub.50 (nM) Max Signal SiMa GFP 0.0812 .+-. 0.010 22,733,787 SiMa FGFR2 0.0459 .+-. 0.003 26,136,578 SiMa FGFR3 0.0377 .+-. 0.006 24,326,271 PC-12 GFP 3.3362 .+-. 1.881 26,956,063 PC-12 FGFR2 0.3429 .+-. 0.059 25,376,114 PC-12 FGFR3 0.2634 .+-. 0.026 24,102,459 Neuro-2a GFP 61.80 .+-. 9.710 4,605,974 Neuro-2a FGFR2 31.59 .+-. 8.800 23,279,765 Neuro-2a FGFR3 11.55 .+-. 5.240 28,347,413

[0289] Detection for the presence of SNAP-25 cleavage product can also be performed using sandwich ELISA as described in Examples VI-X.

Sequence CWU 1

1

14811296PRTClostridium botulinum 1Met Pro Phe Val Asn Lys Gln Phe Asn Tyr Lys Asp Pro Val Asn Gly1 5 10 15Val Asp Ile Ala Tyr Ile Lys Ile Pro Asn Ala Gly Gln Met Gln Pro 20 25 30Val Lys Ala Phe Lys Ile His Asn Lys Ile Trp Val Ile Pro Glu Arg 35 40 45Asp Thr Phe Thr Asn Pro Glu Glu Gly Asp Leu Asn Pro Pro Pro Glu 50 55 60Ala Lys Gln Val Pro Val Ser Tyr Tyr Asp Ser Thr Tyr Leu Ser Thr65 70 75 80Asp Asn Glu Lys Asp Asn Tyr Leu Lys Gly Val Thr Lys Leu Phe Glu 85 90 95Arg Ile Tyr Ser Thr Asp Leu Gly Arg Met Leu Leu Thr Ser Ile Val 100 105 110Arg Gly Ile Pro Phe Trp Gly Gly Ser Thr Ile Asp Thr Glu Leu Lys 115 120 125Val Ile Asp Thr Asn Cys Ile Asn Val Ile Gln Pro Asp Gly Ser Tyr 130 135 140Arg Ser Glu Glu Leu Asn Leu Val Ile Ile Gly Pro Ser Ala Asp Ile145 150 155 160Ile Gln Phe Glu Cys Lys Ser Phe Gly His Glu Val Leu Asn Leu Thr 165 170 175Arg Asn Gly Tyr Gly Ser Thr Gln Tyr Ile Arg Phe Ser Pro Asp Phe 180 185 190Thr Phe Gly Phe Glu Glu Ser Leu Glu Val Asp Thr Asn Pro Leu Leu 195 200 205Gly Ala Gly Lys Phe Ala Thr Asp Pro Ala Val Thr Leu Ala His Glu 210 215 220Leu Ile His Ala Gly His Arg Leu Tyr Gly Ile Ala Ile Asn Pro Asn225 230 235 240Arg Val Phe Lys Val Asn Thr Asn Ala Tyr Tyr Glu Met Ser Gly Leu 245 250 255Glu Val Ser Phe Glu Glu Leu Arg Thr Phe Gly Gly His Asp Ala Lys 260 265 270Phe Ile Asp Ser Leu Gln Glu Asn Glu Phe Arg Leu Tyr Tyr Tyr Asn 275 280 285Lys Phe Lys Asp Ile Ala Ser Thr Leu Asn Lys Ala Lys Ser Ile Val 290 295 300Gly Thr Thr Ala Ser Leu Gln Tyr Met Lys Asn Val Phe Lys Glu Lys305 310 315 320Tyr Leu Leu Ser Glu Asp Thr Ser Gly Lys Phe Ser Val Asp Lys Leu 325 330 335Lys Phe Asp Lys Leu Tyr Lys Met Leu Thr Glu Ile Tyr Thr Glu Asp 340 345 350Asn Phe Val Lys Phe Phe Lys Val Leu Asn Arg Lys Thr Tyr Leu Asn 355 360 365Phe Asp Lys Ala Val Phe Lys Ile Asn Ile Val Pro Lys Val Asn Tyr 370 375 380Thr Ile Tyr Asp Gly Phe Asn Leu Arg Asn Thr Asn Leu Ala Ala Asn385 390 395 400Phe Asn Gly Gln Asn Thr Glu Ile Asn Asn Met Asn Phe Thr Lys Leu 405 410 415Lys Asn Phe Thr Gly Leu Phe Glu Phe Tyr Lys Leu Leu Cys Val Arg 420 425 430Gly Ile Ile Thr Ser Lys Thr Lys Ser Leu Asp Lys Gly Tyr Asn Lys 435 440 445Ala Leu Asn Asp Leu Cys Ile Lys Val Asn Asn Trp Asp Leu Phe Phe 450 455 460Ser Pro Ser Glu Asp Asn Phe Thr Asn Asp Leu Asn Lys Gly Glu Glu465 470 475 480Ile Thr Ser Asp Thr Asn Ile Glu Ala Ala Glu Glu Asn Ile Ser Leu 485 490 495Asp Leu Ile Gln Gln Tyr Tyr Leu Thr Phe Asn Phe Asp Asn Glu Pro 500 505 510Glu Asn Ile Ser Ile Glu Asn Leu Ser Ser Asp Ile Ile Gly Gln Leu 515 520 525Glu Leu Met Pro Asn Ile Glu Arg Phe Pro Asn Gly Lys Lys Tyr Glu 530 535 540Leu Asp Lys Tyr Thr Met Phe His Tyr Leu Arg Ala Gln Glu Phe Glu545 550 555 560His Gly Lys Ser Arg Ile Ala Leu Thr Asn Ser Val Asn Glu Ala Leu 565 570 575Leu Asn Pro Ser Arg Val Tyr Thr Phe Phe Ser Ser Asp Tyr Val Lys 580 585 590Lys Val Asn Lys Ala Thr Glu Ala Ala Met Phe Leu Gly Trp Val Glu 595 600 605Gln Leu Val Tyr Asp Phe Thr Asp Glu Thr Ser Glu Val Ser Thr Thr 610 615 620Asp Lys Ile Ala Asp Ile Thr Ile Ile Ile Pro Tyr Ile Gly Pro Ala625 630 635 640Leu Asn Ile Gly Asn Met Leu Tyr Lys Asp Asp Phe Val Gly Ala Leu 645 650 655Ile Phe Ser Gly Ala Val Ile Leu Leu Glu Phe Ile Pro Glu Ile Ala 660 665 670Ile Pro Val Leu Gly Thr Phe Ala Leu Val Ser Tyr Ile Ala Asn Lys 675 680 685Val Leu Thr Val Gln Thr Ile Asp Asn Ala Leu Ser Lys Arg Asn Glu 690 695 700Lys Trp Asp Glu Val Tyr Lys Tyr Ile Val Thr Asn Trp Leu Ala Lys705 710 715 720Val Asn Thr Gln Ile Asp Leu Ile Arg Lys Lys Met Lys Glu Ala Leu 725 730 735Glu Asn Gln Ala Glu Ala Thr Lys Ala Ile Ile Asn Tyr Gln Tyr Asn 740 745 750Gln Tyr Thr Glu Glu Glu Lys Asn Asn Ile Asn Phe Asn Ile Asp Asp 755 760 765Leu Ser Ser Lys Leu Asn Glu Ser Ile Asn Lys Ala Met Ile Asn Ile 770 775 780Asn Lys Phe Leu Asn Gln Cys Ser Val Ser Tyr Leu Met Asn Ser Met785 790 795 800Ile Pro Tyr Gly Val Lys Arg Leu Glu Asp Phe Asp Ala Ser Leu Lys 805 810 815Asp Ala Leu Leu Lys Tyr Ile Tyr Asp Asn Arg Gly Thr Leu Ile Gly 820 825 830Gln Val Asp Arg Leu Lys Asp Lys Val Asn Asn Thr Leu Ser Thr Asp 835 840 845Ile Pro Phe Gln Leu Ser Lys Tyr Val Asp Asn Gln Arg Leu Leu Ser 850 855 860Thr Phe Thr Glu Tyr Ile Lys Asn Ile Ile Asn Thr Ser Ile Leu Asn865 870 875 880Leu Arg Tyr Glu Ser Asn His Leu Ile Asp Leu Ser Arg Tyr Ala Ser 885 890 895Lys Ile Asn Ile Gly Ser Lys Val Asn Phe Asp Pro Ile Asp Lys Asn 900 905 910Gln Ile Gln Leu Phe Asn Leu Glu Ser Ser Lys Ile Glu Val Ile Leu 915 920 925Lys Asn Ala Ile Val Tyr Asn Ser Met Tyr Glu Asn Phe Ser Thr Ser 930 935 940Phe Trp Ile Arg Ile Pro Lys Tyr Phe Asn Ser Ile Ser Leu Asn Asn945 950 955 960Glu Tyr Thr Ile Ile Asn Cys Met Glu Asn Asn Ser Gly Trp Lys Val 965 970 975Ser Leu Asn Tyr Gly Glu Ile Ile Trp Thr Leu Gln Asp Thr Gln Glu 980 985 990Ile Lys Gln Arg Val Val Phe Lys Tyr Ser Gln Met Ile Asn Ile Ser 995 1000 1005Asp Tyr Ile Asn Arg Trp Ile Phe Val Thr Ile Thr Asn Asn Arg Leu 1010 1015 1020Asn Asn Ser Lys Ile Tyr Ile Asn Gly Arg Leu Ile Asp Gln Lys Pro1025 1030 1035 1040Ile Ser Asn Leu Gly Asn Ile His Ala Ser Asn Asn Ile Met Phe Lys 1045 1050 1055Leu Asp Gly Cys Arg Asp Thr His Arg Tyr Ile Trp Ile Lys Tyr Phe 1060 1065 1070Asn Leu Phe Asp Lys Glu Leu Asn Glu Lys Glu Ile Lys Asp Leu Tyr 1075 1080 1085Asp Asn Gln Ser Asn Ser Gly Ile Leu Lys Asp Phe Trp Gly Asp Tyr 1090 1095 1100Leu Gln Tyr Asp Lys Pro Tyr Tyr Met Leu Asn Leu Tyr Asp Pro Asn1105 1110 1115 1120Lys Tyr Val Asp Val Asn Asn Val Gly Ile Arg Gly Tyr Met Tyr Leu 1125 1130 1135Lys Gly Pro Arg Gly Ser Val Met Thr Thr Asn Ile Tyr Leu Asn Ser 1140 1145 1150Ser Leu Tyr Arg Gly Thr Lys Phe Ile Ile Lys Lys Tyr Ala Ser Gly 1155 1160 1165Asn Lys Asp Asn Ile Val Arg Asn Asn Asp Arg Val Tyr Ile Asn Val 1170 1175 1180Val Val Lys Asn Lys Glu Tyr Arg Leu Ala Thr Asn Ala Ser Gln Ala1185 1190 1195 1200Gly Val Glu Lys Ile Leu Ser Ala Leu Glu Ile Pro Asp Val Gly Asn 1205 1210 1215Leu Ser Gln Val Val Val Met Lys Ser Lys Asn Asp Gln Gly Ile Thr 1220 1225 1230Asn Lys Cys Lys Met Asn Leu Gln Asp Asn Asn Gly Asn Asp Ile Gly 1235 1240 1245Phe Ile Gly Phe His Gln Phe Asn Asn Ile Ala Lys Leu Val Ala Ser 1250 1255 1260Asn Trp Tyr Asn Arg Gln Ile Glu Arg Ser Ser Arg Thr Leu Gly Cys1265 1270 1275 1280Ser Trp Glu Phe Ile Pro Val Asp Asp Gly Trp Gly Glu Arg Pro Leu 1285 1290 129521296PRTClostridium botulinum 2Met Pro Phe Val Asn Lys Gln Phe Asn Tyr Lys Asp Pro Val Asn Gly1 5 10 15Val Asp Ile Ala Tyr Ile Lys Ile Pro Asn Ala Gly Gln Met Gln Pro 20 25 30Val Lys Ala Phe Lys Ile His Asn Lys Ile Trp Val Ile Pro Glu Arg 35 40 45Asp Thr Phe Thr Asn Pro Glu Glu Gly Asp Leu Asn Pro Pro Pro Glu 50 55 60Ala Lys Gln Val Pro Val Ser Tyr Tyr Asp Ser Thr Tyr Leu Ser Thr65 70 75 80Asp Asn Glu Lys Asp Asn Tyr Leu Lys Gly Val Thr Lys Leu Phe Glu 85 90 95Arg Ile Tyr Ser Thr Asp Leu Gly Arg Met Leu Leu Thr Ser Ile Val 100 105 110Arg Gly Ile Pro Phe Trp Gly Gly Ser Thr Ile Asp Thr Glu Leu Lys 115 120 125Val Ile Asp Thr Asn Cys Ile Asn Val Ile Gln Pro Asp Gly Ser Tyr 130 135 140Arg Ser Glu Glu Leu Asn Leu Val Ile Ile Gly Pro Ser Ala Asp Ile145 150 155 160Ile Gln Phe Glu Cys Lys Ser Phe Gly His Asp Val Leu Asn Leu Thr 165 170 175Arg Asn Gly Tyr Gly Ser Thr Gln Tyr Ile Arg Phe Ser Pro Asp Phe 180 185 190Thr Phe Gly Phe Glu Glu Ser Leu Glu Val Asp Thr Asn Pro Leu Leu 195 200 205Gly Ala Gly Lys Phe Ala Thr Asp Pro Ala Val Thr Leu Ala His Glu 210 215 220Leu Ile His Ala Glu His Arg Leu Tyr Gly Ile Ala Ile Asn Pro Asn225 230 235 240Arg Val Phe Lys Val Asn Thr Asn Ala Tyr Tyr Glu Met Ser Gly Leu 245 250 255Glu Val Ser Phe Glu Glu Leu Arg Thr Phe Gly Gly His Asp Ala Lys 260 265 270Phe Ile Asp Ser Leu Gln Glu Asn Glu Phe Arg Leu Tyr Tyr Tyr Asn 275 280 285Lys Phe Lys Asp Val Ala Ser Thr Leu Asn Lys Ala Lys Ser Ile Ile 290 295 300Gly Thr Thr Ala Ser Leu Gln Tyr Met Lys Asn Val Phe Lys Glu Lys305 310 315 320Tyr Leu Leu Ser Glu Asp Thr Ser Gly Lys Phe Ser Val Asp Lys Leu 325 330 335Lys Phe Asp Lys Leu Tyr Lys Met Leu Thr Glu Ile Tyr Thr Glu Asp 340 345 350Asn Phe Val Asn Phe Phe Lys Val Ile Asn Arg Lys Thr Tyr Leu Asn 355 360 365Phe Asp Lys Ala Val Phe Arg Ile Asn Ile Val Pro Asp Glu Asn Tyr 370 375 380Thr Ile Lys Asp Gly Phe Asn Leu Lys Gly Ala Asn Leu Ser Thr Asn385 390 395 400Phe Asn Gly Gln Asn Thr Glu Ile Asn Ser Arg Asn Phe Thr Arg Leu 405 410 415Lys Asn Phe Thr Gly Leu Phe Glu Phe Tyr Lys Leu Leu Cys Val Arg 420 425 430Gly Ile Ile Pro Phe Lys Thr Lys Ser Leu Asp Glu Gly Tyr Asn Lys 435 440 445Ala Leu Asn Asp Leu Cys Ile Lys Val Asn Asn Trp Asp Leu Phe Phe 450 455 460Ser Pro Ser Glu Asp Asn Phe Thr Asn Asp Leu Asp Lys Val Glu Glu465 470 475 480Ile Thr Ala Asp Thr Asn Ile Glu Ala Ala Glu Glu Asn Ile Ser Leu 485 490 495Asp Leu Ile Gln Gln Tyr Tyr Leu Thr Phe Asp Phe Asp Asn Glu Pro 500 505 510Glu Asn Ile Ser Ile Glu Asn Leu Ser Ser Asp Ile Ile Gly Gln Leu 515 520 525Glu Pro Met Pro Asn Ile Glu Arg Phe Pro Asn Gly Lys Lys Tyr Glu 530 535 540Leu Asp Lys Tyr Thr Met Phe His Tyr Leu Arg Ala Gln Glu Phe Glu545 550 555 560His Gly Asp Ser Arg Ile Ile Leu Thr Asn Ser Ala Glu Glu Ala Leu 565 570 575Leu Lys Pro Asn Val Ala Tyr Thr Phe Phe Ser Ser Lys Tyr Val Lys 580 585 590Lys Ile Asn Lys Ala Val Glu Ala Phe Met Phe Leu Asn Trp Ala Glu 595 600 605Glu Leu Val Tyr Asp Phe Thr Asp Glu Thr Asn Glu Val Thr Thr Met 610 615 620Asp Lys Ile Ala Asp Ile Thr Ile Ile Val Pro Tyr Ile Gly Pro Ala625 630 635 640Leu Asn Ile Gly Asn Met Leu Ser Lys Gly Glu Phe Val Glu Ala Ile 645 650 655Ile Phe Thr Gly Val Val Ala Met Leu Glu Phe Ile Pro Glu Tyr Ala 660 665 670Leu Pro Val Phe Gly Thr Phe Ala Ile Val Ser Tyr Ile Ala Asn Lys 675 680 685Val Leu Thr Val Gln Thr Ile Asn Asn Ala Leu Ser Lys Arg Asn Glu 690 695 700Lys Trp Asp Glu Val Tyr Lys Tyr Thr Val Thr Asn Trp Leu Ala Lys705 710 715 720Val Asn Thr Gln Ile Asp Leu Ile Arg Glu Lys Met Lys Lys Ala Leu 725 730 735Glu Asn Gln Ala Glu Ala Thr Lys Ala Ile Ile Asn Tyr Gln Tyr Asn 740 745 750Gln Tyr Thr Glu Glu Glu Lys Asn Asn Ile Asn Phe Asn Ile Asp Asp 755 760 765Leu Ser Ser Lys Leu Asn Glu Ser Ile Asn Ser Ala Met Ile Asn Ile 770 775 780Asn Lys Phe Leu Asp Gln Cys Ser Val Ser Tyr Leu Met Asn Ser Met785 790 795 800Ile Pro Tyr Ala Val Lys Arg Leu Lys Asp Phe Asp Ala Ser Val Arg 805 810 815Asp Val Leu Leu Lys Tyr Ile Tyr Asp Asn Arg Gly Thr Leu Val Leu 820 825 830Gln Val Asp Arg Leu Lys Asp Glu Val Asn Asn Thr Leu Ser Ala Asp 835 840 845Ile Pro Phe Gln Leu Ser Lys Tyr Val Asp Asn Lys Lys Leu Leu Ser 850 855 860Thr Phe Thr Glu Tyr Ile Lys Asn Ile Val Asn Thr Ser Ile Leu Ser865 870 875 880Ile Val Tyr Lys Lys Asp Asp Leu Ile Asp Leu Ser Arg Tyr Gly Ala 885 890 895Lys Ile Asn Ile Gly Asp Arg Val Tyr Tyr Asp Ser Ile Asp Lys Asn 900 905 910Gln Ile Lys Leu Ile Asn Leu Glu Ser Ser Thr Ile Glu Val Ile Leu 915 920 925Lys Asn Ala Ile Val Tyr Asn Ser Met Tyr Glu Asn Phe Ser Thr Ser 930 935 940Phe Trp Ile Lys Ile Pro Lys Tyr Phe Ser Lys Ile Asn Leu Asn Asn945 950 955 960Glu Tyr Thr Ile Ile Asn Cys Ile Glu Asn Asn Ser Gly Trp Lys Val 965 970 975Ser Leu Asn Tyr Gly Glu Ile Ile Trp Thr Leu Gln Asp Asn Lys Gln 980 985 990Asn Ile Gln Arg Val Val Phe Lys Tyr Ser Gln Met Val Asn Ile Ser 995 1000 1005Asp Tyr Ile Asn Arg Trp Ile Phe Val Thr Ile Thr Asn Asn Arg Leu 1010 1015 1020Thr Lys Ser Lys Ile Tyr Ile Asn Gly Arg Leu Ile Asp Gln Lys Pro1025 1030 1035 1040Ile Ser Asn Leu Gly Asn Ile His Ala Ser Asn Lys Ile Met Phe Lys 1045 1050 1055Leu Asp Gly Cys Arg Asp Pro Arg Arg Tyr Ile Met Ile Lys Tyr Phe 1060 1065 1070Asn Leu Phe Asp Lys Glu Leu Asn Glu Lys Glu Ile Lys Asp Leu Tyr 1075 1080 1085Asp Ser Gln Ser Asn Ser Gly Ile Leu Lys Asp Phe Trp Gly Asn Tyr 1090 1095 1100Leu Gln Tyr Asp Lys Pro Tyr Tyr Met Leu Asn Leu Phe Asp Pro Asn1105 1110 1115 1120Lys Tyr Val Asp Val Asn Asn Ile Gly Ile Arg Gly Tyr Met Tyr Leu 1125 1130 1135Lys Gly Pro Arg Gly Ser Val Val Thr Thr Asn Ile Tyr Leu Asn Ser 1140 1145 1150Thr Leu Tyr Glu Gly Thr Lys Phe Ile Ile Lys Lys Tyr Ala Ser Gly 1155 1160

1165Asn Glu Asp Asn Ile Val Arg Asn Asn Asp Arg Val Tyr Ile Asn Val 1170 1175 1180Val Val Lys Asn Lys Glu Tyr Arg Leu Ala Thr Asn Ala Ser Gln Ala1185 1190 1195 1200Gly Val Glu Lys Ile Leu Ser Ala Leu Glu Ile Pro Asp Val Gly Asn 1205 1210 1215Leu Ser Gln Val Val Val Met Lys Ser Lys Asp Asp Gln Gly Ile Arg 1220 1225 1230Asn Lys Cys Lys Met Asn Leu Gln Asp Asn Asn Gly Asn Asp Ile Gly 1235 1240 1245Phe Ile Gly Phe His Leu Tyr Asp Asn Ile Ala Lys Leu Val Ala Ser 1250 1255 1260Asn Trp Tyr Asn Arg Gln Val Gly Lys Ala Ser Arg Thr Phe Gly Cys1265 1270 1275 1280Ser Trp Glu Phe Ile Pro Val Asp Asp Gly Trp Gly Glu Ser Ser Leu 1285 1290 129531292PRTClostridium botulinum 3Met Pro Phe Val Asn Lys Pro Phe Asn Tyr Arg Asp Pro Gly Asn Gly1 5 10 15Val Asp Ile Ala Tyr Ile Lys Ile Pro Asn Ala Gly Gln Met Gln Pro 20 25 30Val Lys Ala Phe Lys Ile His Glu Gly Val Trp Val Ile Pro Glu Arg 35 40 45Asp Thr Phe Thr Asn Pro Glu Glu Gly Asp Leu Asn Pro Pro Pro Glu 50 55 60Ala Lys Gln Val Pro Val Ser Tyr Tyr Asp Ser Thr Tyr Leu Ser Thr65 70 75 80Asp Asn Glu Lys Asp Asn Tyr Leu Lys Gly Val Ile Lys Leu Phe Asp 85 90 95Arg Ile Tyr Ser Thr Gly Leu Gly Arg Met Leu Leu Ser Phe Ile Val 100 105 110Lys Gly Ile Pro Phe Trp Gly Gly Ser Thr Ile Asp Thr Glu Leu Lys 115 120 125Val Ile Asp Thr Asn Cys Ile Asn Val Ile Glu Pro Gly Gly Ser Tyr 130 135 140Arg Ser Glu Glu Leu Asn Leu Val Ile Thr Gly Pro Ser Ala Asp Ile145 150 155 160Ile Gln Phe Glu Cys Lys Ser Phe Gly His Asp Val Phe Asn Leu Thr 165 170 175Arg Asn Gly Tyr Gly Ser Thr Gln Tyr Ile Arg Phe Ser Pro Asp Phe 180 185 190Thr Phe Gly Phe Glu Glu Ser Leu Glu Val Asp Thr Asn Pro Leu Leu 195 200 205Gly Ala Gly Thr Phe Ala Thr Asp Pro Ala Val Thr Leu Ala His Glu 210 215 220Leu Ile His Ala Ala His Arg Leu Tyr Gly Ile Ala Ile Asn Pro Asn225 230 235 240Arg Val Leu Lys Val Lys Thr Asn Ala Tyr Tyr Glu Met Ser Gly Leu 245 250 255Glu Val Ser Phe Glu Glu Leu Arg Thr Phe Gly Gly Asn Asp Thr Asn 260 265 270Phe Ile Asp Ser Leu Trp Gln Lys Lys Phe Ser Arg Asp Ala Tyr Asp 275 280 285Asn Leu Gln Asn Ile Ala Arg Ile Leu Asn Glu Ala Lys Thr Ile Val 290 295 300Gly Thr Thr Thr Pro Leu Gln Tyr Met Lys Asn Ile Phe Ile Arg Lys305 310 315 320Tyr Phe Leu Ser Glu Asp Ala Ser Gly Lys Ile Ser Val Asn Lys Ala 325 330 335Ala Phe Lys Glu Phe Tyr Arg Val Leu Thr Arg Gly Phe Thr Glu Leu 340 345 350Glu Phe Val Asn Pro Phe Lys Val Ile Asn Arg Lys Thr Tyr Leu Asn 355 360 365Phe Asp Lys Ala Val Phe Arg Ile Asn Ile Val Pro Asp Glu Asn Tyr 370 375 380Thr Ile Asn Glu Gly Phe Asn Leu Glu Gly Ala Asn Ser Asn Gly Gln385 390 395 400Asn Thr Glu Ile Asn Ser Arg Asn Phe Thr Arg Leu Lys Asn Phe Thr 405 410 415Gly Leu Phe Glu Phe Tyr Lys Leu Leu Cys Val Arg Gly Ile Ile Pro 420 425 430Phe Lys Thr Lys Ser Leu Asp Glu Gly Tyr Asn Lys Ala Leu Asn Tyr 435 440 445Leu Cys Ile Lys Val Asn Asn Trp Asp Leu Phe Phe Ser Pro Ser Glu 450 455 460Asp Asn Phe Thr Asn Asp Leu Asp Lys Val Glu Glu Ile Thr Ala Asp465 470 475 480Thr Asn Ile Glu Ala Ala Glu Glu Asn Ile Ser Ser Asp Leu Ile Gln 485 490 495Gln Tyr Tyr Leu Thr Phe Asp Phe Asp Asn Glu Pro Glu Asn Ile Ser 500 505 510Ile Glu Asn Leu Ser Ser Asp Ile Ile Gly Gln Leu Glu Pro Met Pro 515 520 525Asn Ile Glu Arg Phe Pro Asn Gly Lys Lys Tyr Glu Leu Asp Lys Tyr 530 535 540Thr Met Phe His Tyr Leu Arg Ala Gln Glu Phe Glu His Gly Asp Ser545 550 555 560Arg Ile Ile Leu Thr Asn Ser Ala Glu Glu Ala Leu Leu Lys Pro Asn 565 570 575Val Ala Tyr Thr Phe Phe Ser Ser Lys Tyr Val Lys Lys Ile Asn Lys 580 585 590Ala Val Glu Ala Val Ile Phe Leu Ser Trp Ala Glu Glu Leu Val Tyr 595 600 605Asp Phe Thr Asp Glu Thr Asn Glu Val Thr Thr Met Asp Lys Ile Ala 610 615 620Asp Ile Thr Ile Ile Val Pro Tyr Ile Gly Pro Ala Leu Asn Ile Gly625 630 635 640Asn Met Val Ser Lys Gly Glu Phe Val Glu Ala Ile Leu Phe Thr Gly 645 650 655Val Val Ala Leu Leu Glu Phe Ile Pro Glu Tyr Ser Leu Pro Val Phe 660 665 670Gly Thr Phe Ala Ile Val Ser Tyr Ile Ala Asn Lys Val Leu Thr Val 675 680 685Gln Thr Ile Asn Asn Ala Leu Ser Lys Arg Asn Glu Lys Trp Asp Glu 690 695 700Val Tyr Lys Tyr Thr Val Thr Asn Trp Leu Ala Lys Val Asn Thr Gln705 710 715 720Ile Asp Leu Ile Arg Glu Lys Met Lys Lys Ala Leu Glu Asn Gln Ala 725 730 735Glu Ala Thr Arg Ala Ile Ile Asn Tyr Gln Tyr Asn Gln Tyr Thr Glu 740 745 750Glu Glu Lys Asn Asn Ile Asn Phe Asn Ile Asp Asp Leu Ser Ser Lys 755 760 765Leu Asn Arg Ser Ile Asn Arg Ala Met Ile Asn Ile Asn Lys Phe Leu 770 775 780Asp Gln Cys Ser Val Ser Tyr Leu Met Asn Ser Met Ile Pro Tyr Ala785 790 795 800Val Lys Arg Leu Lys Asp Phe Asp Ala Ser Val Arg Asp Val Leu Leu 805 810 815Lys Tyr Ile Tyr Asp Asn Arg Gly Thr Leu Ile Leu Gln Val Asp Arg 820 825 830Leu Lys Asp Glu Val Asn Asn Thr Leu Ser Ala Asp Ile Pro Phe Gln 835 840 845Leu Ser Lys Tyr Val Asn Asp Lys Lys Leu Leu Ser Thr Phe Thr Glu 850 855 860Tyr Ile Lys Asn Ile Val Asn Thr Ser Ile Leu Ser Ile Val Tyr Lys865 870 875 880Lys Asp Asp Leu Ile Asp Leu Ser Arg Tyr Gly Ala Lys Ile Asn Ile 885 890 895Gly Asp Arg Val Tyr Tyr Asp Ser Ile Asp Lys Asn Gln Ile Lys Leu 900 905 910Ile Asn Leu Glu Ser Ser Thr Ile Glu Val Ile Leu Lys Asn Ala Ile 915 920 925Val Tyr Asn Ser Met Tyr Glu Asn Phe Ser Thr Ser Phe Trp Ile Lys 930 935 940Ile Pro Lys Tyr Phe Ser Lys Ile Asn Leu Asn Asn Glu Tyr Thr Ile945 950 955 960Ile Asn Cys Ile Glu Asn Asn Ser Gly Trp Lys Val Ser Leu Asn Tyr 965 970 975Gly Glu Ile Ile Trp Thr Leu Gln Asp Asn Lys Gln Asn Ile Gln Arg 980 985 990Val Val Phe Lys Tyr Ser Gln Met Val Asn Ile Ser Asp Tyr Ile Asn 995 1000 1005Arg Trp Met Phe Val Thr Ile Thr Asn Asn Arg Leu Thr Lys Ser Lys 1010 1015 1020Ile Tyr Ile Asn Gly Arg Leu Ile Asp Gln Lys Pro Ile Ser Asn Leu1025 1030 1035 1040Gly Asn Ile His Ala Ser Asn Lys Ile Met Phe Lys Leu Asp Gly Cys 1045 1050 1055Arg Asp Pro Arg Arg Tyr Ile Met Ile Lys Tyr Phe Asn Leu Phe Asp 1060 1065 1070Lys Glu Leu Asn Glu Lys Glu Ile Lys Asp Leu Tyr Asp Ser Gln Ser 1075 1080 1085Asn Pro Gly Ile Leu Lys Asp Phe Trp Gly Asn Tyr Leu Gln Tyr Asp 1090 1095 1100Lys Pro Tyr Tyr Met Leu Asn Leu Phe Asp Pro Asn Lys Tyr Val Asp1105 1110 1115 1120Val Asn Asn Ile Gly Ile Arg Gly Tyr Met Tyr Leu Lys Gly Pro Arg 1125 1130 1135Gly Ser Val Met Thr Thr Asn Ile Tyr Leu Asn Ser Thr Leu Tyr Met 1140 1145 1150Gly Thr Lys Phe Ile Ile Lys Lys Tyr Ala Ser Gly Asn Glu Asp Asn 1155 1160 1165Ile Val Arg Asn Asn Asp Arg Val Tyr Ile Asn Val Val Val Lys Asn 1170 1175 1180Lys Glu Tyr Arg Leu Ala Thr Asn Ala Ser Gln Ala Gly Val Glu Lys1185 1190 1195 1200Ile Leu Ser Ala Leu Glu Ile Pro Asp Val Gly Asn Leu Ser Gln Val 1205 1210 1215Val Val Met Lys Ser Lys Asp Asp Gln Gly Ile Arg Asn Lys Cys Lys 1220 1225 1230Met Asn Leu Gln Asp Asn Asn Gly Asn Asp Ile Gly Phe Val Gly Phe 1235 1240 1245His Leu Tyr Asp Asn Ile Ala Lys Leu Val Ala Ser Asn Trp Tyr Asn 1250 1255 1260Arg Gln Val Gly Lys Ala Ser Arg Thr Phe Gly Cys Ser Trp Glu Phe1265 1270 1275 1280Ile Pro Val Asp Asp Gly Trp Gly Glu Ser Ser Leu 1285 129041296PRTClostridium botulinum 4Met Pro Leu Val Asn Gln Gln Ile Asn Tyr Tyr Asp Pro Val Asn Gly1 5 10 15Val Asp Ile Ala Tyr Ile Lys Ile Pro Asn Ala Gly Lys Met Gln Pro 20 25 30Val Lys Ala Phe Lys Ile His Asn Lys Val Trp Val Ile Pro Glu Arg 35 40 45Asp Ile Phe Thr Asn Pro Glu Glu Val Asp Leu Asn Pro Pro Pro Glu 50 55 60Ala Lys Gln Val Pro Ile Ser Tyr Tyr Asp Ser Ala Tyr Leu Ser Thr65 70 75 80Asp Asn Glu Lys Asp Asn Tyr Leu Lys Gly Val Ile Lys Leu Phe Glu 85 90 95Arg Ile Tyr Ser Thr Asp Leu Gly Arg Met Leu Leu Ile Ser Ile Val 100 105 110Arg Gly Ile Pro Phe Trp Gly Gly Gly Lys Ile Asp Thr Glu Leu Lys 115 120 125Val Ile Asp Thr Asn Cys Ile Asn Ile Ile Gln Leu Asp Asp Ser Tyr 130 135 140Arg Ser Glu Glu Leu Asn Leu Ala Ile Ile Gly Pro Ser Ala Asn Ile145 150 155 160Ile Glu Ser Gln Cys Ser Ser Phe Arg Asp Asp Val Leu Asn Leu Thr 165 170 175Arg Asn Gly Tyr Gly Ser Thr Gln Tyr Ile Arg Phe Ser Pro Asp Phe 180 185 190Thr Val Gly Phe Glu Glu Ser Leu Glu Val Asp Thr Asn Pro Leu Leu 195 200 205Gly Ala Gly Lys Phe Ala Gln Asp Pro Ala Val Ala Leu Ala His Glu 210 215 220Leu Ile His Ala Glu His Arg Leu Tyr Gly Ile Ala Ile Asn Thr Asn225 230 235 240Arg Val Phe Lys Val Asn Thr Asn Ala Tyr Tyr Glu Met Ala Gly Leu 245 250 255Glu Val Ser Leu Glu Glu Leu Ile Thr Phe Gly Gly Asn Asp Ala Lys 260 265 270Phe Ile Asp Ser Leu Gln Lys Lys Glu Phe Ser Leu Tyr Tyr Tyr Asn 275 280 285Lys Phe Lys Asp Ile Ala Ser Thr Leu Asn Lys Ala Lys Ser Ile Val 290 295 300Gly Thr Thr Ala Ser Leu Gln Tyr Met Lys Asn Val Phe Lys Glu Lys305 310 315 320Tyr Leu Leu Ser Glu Asp Ala Thr Gly Lys Phe Leu Val Asp Arg Leu 325 330 335Lys Phe Asp Glu Leu Tyr Lys Leu Leu Thr Glu Ile Tyr Thr Glu Asp 340 345 350Asn Phe Val Lys Phe Phe Lys Val Leu Asn Arg Lys Thr Tyr Leu Asn 355 360 365Phe Asp Lys Ala Val Phe Lys Ile Asn Ile Val Pro Asp Val Asn Tyr 370 375 380Thr Ile His Asp Gly Phe Asn Leu Arg Asn Thr Asn Leu Ala Ala Asn385 390 395 400Phe Asn Gly Gln Asn Ile Glu Ile Asn Asn Lys Asn Phe Asp Lys Leu 405 410 415Lys Asn Phe Thr Gly Leu Phe Glu Phe Tyr Lys Leu Leu Cys Val Arg 420 425 430Gly Ile Ile Thr Ser Lys Thr Lys Ser Leu Asp Glu Gly Tyr Asn Lys 435 440 445Ala Leu Asn Glu Leu Cys Ile Lys Val Asn Asn Trp Asp Leu Phe Phe 450 455 460Ser Pro Ser Glu Asp Asn Phe Thr Asn Asp Leu Asp Lys Val Glu Glu465 470 475 480Ile Thr Ser Asp Thr Asn Ile Glu Ala Ala Glu Glu Asn Ile Ser Leu 485 490 495Asp Leu Ile Gln Gln Tyr Tyr Leu Asn Phe Asn Phe Asp Asn Glu Pro 500 505 510Glu Asn Thr Ser Ile Glu Asn Leu Ser Ser Asp Ile Ile Gly Gln Leu 515 520 525Glu Pro Met Pro Asn Ile Glu Arg Phe Pro Asn Gly Lys Lys Tyr Glu 530 535 540Leu Asn Lys Tyr Thr Met Phe His Tyr Leu Arg Ala Gln Glu Phe Lys545 550 555 560His Ser Asn Ser Arg Ile Ile Leu Thr Asn Ser Ala Lys Glu Ala Leu 565 570 575Leu Lys Pro Asn Ile Val Tyr Thr Phe Phe Ser Ser Lys Tyr Ile Lys 580 585 590Ala Ile Asn Lys Ala Val Glu Ala Val Thr Phe Val Asn Trp Ile Glu 595 600 605Asn Leu Val Tyr Asp Phe Thr Asp Glu Thr Asn Glu Val Ser Thr Met 610 615 620Asp Lys Ile Ala Asp Ile Thr Ile Val Ile Pro Tyr Ile Gly Pro Ala625 630 635 640Leu Asn Ile Gly Asn Met Ile Tyr Lys Gly Glu Phe Val Glu Ala Ile 645 650 655Ile Phe Ser Gly Ala Val Ile Leu Leu Glu Ile Val Pro Glu Ile Ala 660 665 670Leu Pro Val Leu Gly Thr Phe Ala Leu Val Ser Tyr Val Ser Asn Lys 675 680 685Val Leu Thr Val Gln Thr Ile Asp Asn Ala Leu Ser Lys Arg Asn Glu 690 695 700Lys Trp Asp Glu Val Tyr Lys Tyr Ile Val Thr Asn Trp Leu Ala Ile705 710 715 720Val Asn Thr Gln Ile Asn Leu Ile Arg Glu Lys Met Lys Lys Ala Leu 725 730 735Glu Asn Gln Ala Glu Ala Thr Lys Ala Ile Ile Asn Tyr Gln Tyr Asn 740 745 750Gln Tyr Thr Glu Glu Glu Lys Asn Asn Ile Asn Phe Asn Ile Asp Asp 755 760 765Leu Ser Ser Lys Leu Asn Glu Ser Ile Asn Ser Ala Met Ile Asn Ile 770 775 780Asn Lys Phe Leu Asp Gln Cys Ser Val Ser Tyr Leu Met Asn Ser Met785 790 795 800Ile Pro Tyr Ala Val Lys Arg Leu Lys Asp Phe Asp Ala Ser Val Arg 805 810 815Asp Val Leu Leu Lys Tyr Ile Tyr Asp Asn Arg Gly Thr Leu Ile Gly 820 825 830Gln Val Asn Arg Leu Lys Asp Lys Val Asn Asn Thr Leu Ser Ala Asp 835 840 845Ile Pro Phe Gln Leu Ser Lys Tyr Val Asp Asn Lys Lys Leu Leu Ser 850 855 860Thr Phe Thr Glu Tyr Ile Lys Asn Ile Thr Asn Ala Ser Ile Leu Ser865 870 875 880Ile Val Tyr Lys Asp Asp Asp Leu Ile Asp Leu Ser Arg Tyr Gly Ala 885 890 895Glu Ile Tyr Asn Gly Asp Lys Val Tyr Tyr Asn Ser Ile Asp Lys Asn 900 905 910Gln Ile Arg Leu Ile Asn Leu Glu Ser Ser Thr Ile Glu Val Ile Leu 915 920 925Lys Lys Ala Ile Val Tyr Asn Ser Met Tyr Glu Asn Phe Ser Thr Ser 930 935 940Phe Trp Ile Arg Ile Pro Lys Tyr Phe Asn Ser Ile Ser Leu Asn Asn945 950 955 960Glu Tyr Thr Ile Ile Asn Cys Met Glu Asn Asn Ser Gly Trp Lys Val 965 970 975Ser Leu Asn Tyr Gly Glu Ile Ile Trp Thr Phe Gln Asp Thr Gln Glu 980 985 990Ile Lys Gln Arg Val Val Phe Lys Tyr Ser Gln Met Ile Asn Ile Ser 995 1000 1005Asp Tyr Ile Asn Arg Trp Ile Phe Val Thr Ile Thr Asn Asn Arg Ile 1010 1015 1020Thr Lys Ser Lys Ile Tyr Ile Asn Gly Arg Leu Ile Asp Gln Lys Pro1025 1030 1035

1040Ile Ser Asn Leu Gly Asn Ile His Ala Ser Asn Lys Ile Met Phe Lys 1045 1050 1055Leu Asp Gly Cys Arg Asp Pro His Arg Tyr Ile Val Ile Lys Tyr Phe 1060 1065 1070Asn Leu Phe Asp Lys Glu Leu Ser Glu Lys Glu Ile Lys Asp Leu Tyr 1075 1080 1085Asp Asn Gln Ser Asn Ser Gly Ile Leu Lys Asp Phe Trp Gly Asp Tyr 1090 1095 1100Leu Gln Tyr Asp Lys Ser Tyr Tyr Met Leu Asn Leu Tyr Asp Pro Asn1105 1110 1115 1120Lys Tyr Val Asp Val Asn Asn Val Gly Ile Arg Gly Tyr Met Tyr Leu 1125 1130 1135Lys Gly Pro Arg Asp Asn Val Met Thr Thr Asn Ile Tyr Leu Asn Ser 1140 1145 1150Ser Leu Tyr Met Gly Thr Lys Phe Ile Ile Lys Lys Tyr Ala Ser Gly 1155 1160 1165Asn Lys Asp Asn Ile Val Arg Asn Asn Asp Arg Val Tyr Ile Asn Val 1170 1175 1180Val Val Lys Asn Lys Glu Tyr Arg Leu Ala Thr Asn Ala Ser Gln Ala1185 1190 1195 1200Gly Val Glu Lys Ile Leu Ser Ala Leu Glu Ile Pro Asp Val Gly Asn 1205 1210 1215Leu Ser Gln Val Val Val Met Lys Ser Lys Asn Asp Gln Gly Ile Thr 1220 1225 1230Asn Lys Cys Lys Met Asn Leu Gln Asp Asn Asn Gly Asn Asp Ile Gly 1235 1240 1245Phe Ile Gly Phe His Gln Phe Asn Asn Ile Ala Lys Leu Val Ala Ser 1250 1255 1260Asn Trp Tyr Asn Arg Gln Ile Glu Arg Ser Ser Arg Thr Leu Gly Cys1265 1270 1275 1280Ser Trp Glu Phe Ile Pro Val Asp Asp Gly Trp Arg Glu Arg Pro Leu 1285 1290 12955206PRTHomo sapiens 5Met Ala Glu Asp Ala Asp Met Arg Asn Glu Leu Glu Glu Met Gln Arg1 5 10 15Arg Ala Asp Gln Leu Ala Asp Glu Ser Leu Glu Ser Thr Arg Arg Met 20 25 30Leu Gln Leu Val Glu Glu Ser Lys Asp Ala Gly Ile Arg Thr Leu Val 35 40 45Met Leu Asp Glu Gln Gly Glu Gln Leu Asp Arg Val Glu Glu Gly Met 50 55 60Asn His Ile Asn Gln Asp Met Lys Glu Ala Glu Lys Asn Leu Lys Asp65 70 75 80Leu Gly Lys Cys Cys Gly Leu Phe Ile Cys Pro Cys Asn Lys Leu Lys 85 90 95Ser Ser Asp Ala Tyr Lys Lys Ala Trp Gly Asn Asn Gln Asp Gly Val 100 105 110Val Ala Ser Gln Pro Ala Arg Val Val Asp Glu Arg Glu Gln Met Ala 115 120 125Ile Ser Gly Gly Phe Ile Arg Arg Val Thr Asn Asp Ala Arg Glu Asn 130 135 140Glu Met Asp Glu Asn Leu Glu Gln Val Ser Gly Ile Ile Gly Asn Leu145 150 155 160Arg His Met Ala Leu Asp Met Gly Asn Glu Ile Asp Thr Gln Asn Arg 165 170 175Gln Ile Asp Arg Ile Met Glu Lys Ala Asp Ser Asn Lys Thr Arg Ile 180 185 190Asp Glu Ala Asn Gln Arg Ala Thr Lys Met Leu Gly Ser Gly 195 200 2056206PRTHomo sapiens 6Met Ala Glu Asp Ala Asp Met Arg Asn Glu Leu Glu Glu Met Gln Arg1 5 10 15Arg Ala Asp Gln Leu Ala Asp Glu Ser Leu Glu Ser Thr Arg Arg Met 20 25 30Leu Gln Leu Val Glu Glu Ser Lys Asp Ala Gly Ile Arg Thr Leu Val 35 40 45Met Leu Asp Glu Gln Gly Glu Gln Leu Glu Arg Ile Glu Glu Gly Met 50 55 60Asp Gln Ile Asn Lys Asp Met Lys Glu Ala Glu Lys Asn Leu Thr Asp65 70 75 80Leu Gly Lys Phe Cys Gly Leu Cys Val Cys Pro Cys Asn Lys Leu Lys 85 90 95Ser Ser Asp Ala Tyr Lys Lys Ala Trp Gly Asn Asn Gln Asp Gly Val 100 105 110Val Ala Ser Gln Pro Ala Arg Val Val Asp Glu Arg Glu Gln Met Ala 115 120 125Ile Ser Gly Gly Phe Ile Arg Arg Val Thr Asn Asp Ala Arg Glu Asn 130 135 140Glu Met Asp Glu Asn Leu Glu Gln Val Ser Gly Ile Ile Gly Asn Leu145 150 155 160Arg His Met Ala Leu Asp Met Gly Asn Glu Ile Asp Thr Gln Asn Arg 165 170 175Gln Ile Asp Arg Ile Met Glu Lys Ala Asp Ser Asn Lys Thr Arg Ile 180 185 190Asp Glu Ala Asn Gln Arg Ala Thr Lys Met Leu Gly Ser Gly 195 200 2057206PRTMacaca mulatta 7Met Ala Glu Asp Ala Asp Met Arg Asn Glu Leu Glu Glu Met Gln Arg1 5 10 15Arg Ala Asp Gln Leu Ala Asp Glu Ser Leu Glu Ser Thr Arg Arg Met 20 25 30Leu Gln Leu Val Glu Glu Ser Lys Asp Ala Gly Ile Arg Thr Leu Val 35 40 45Met Leu Asp Glu Gln Gly Glu Gln Leu Glu Arg Ile Glu Glu Gly Met 50 55 60Asp Gln Ile Asn Lys Asp Met Lys Glu Ala Glu Lys Asn Leu Thr Asp65 70 75 80Leu Gly Lys Phe Cys Gly Leu Cys Val Cys Pro Cys Asn Lys Leu Lys 85 90 95Ser Ser Asp Ala Tyr Lys Lys Ala Trp Gly Asn Asn Gln Asp Gly Val 100 105 110Val Ala Ser Gln Pro Ala Arg Val Val Asp Glu Arg Glu Gln Met Ala 115 120 125Ile Ser Gly Gly Phe Ile Arg Arg Val Thr Asn Asp Ala Arg Glu Asn 130 135 140Glu Met Asp Glu Asn Leu Glu Gln Val Ser Gly Ile Ile Gly Asn Leu145 150 155 160Arg His Met Ala Leu Asp Met Gly Asn Glu Ile Asp Thr Gln Asn Arg 165 170 175Gln Ile Asp Arg Ile Met Glu Lys Ala Asp Ser Asn Lys Thr Arg Ile 180 185 190Asp Glu Ala Asn Gln Arg Ala Thr Lys Met Leu Gly Ser Gly 195 200 2058206PRTRattus norvegicus 8Met Ala Glu Asp Ala Asp Met Arg Asn Glu Leu Glu Glu Met Gln Arg1 5 10 15Arg Ala Asp Gln Leu Ala Asp Glu Ser Leu Glu Ser Thr Arg Arg Met 20 25 30Leu Gln Leu Val Glu Glu Ser Lys Asp Ala Gly Ile Arg Thr Leu Val 35 40 45Met Leu Asp Glu Gln Gly Glu Gln Leu Asp Arg Val Glu Glu Gly Met 50 55 60Asn His Ile Asn Gln Asp Met Lys Glu Ala Glu Lys Asn Leu Lys Asp65 70 75 80Leu Gly Lys Cys Cys Gly Leu Phe Ile Cys Pro Cys Asn Lys Leu Lys 85 90 95Ser Ser Asp Ala Tyr Lys Lys Ala Trp Gly Asn Asn Gln Asp Gly Val 100 105 110Val Ala Ser Gln Pro Ala Arg Val Val Asp Glu Arg Glu Gln Met Ala 115 120 125Ile Ser Gly Gly Phe Ile Arg Arg Val Thr Asn Asp Ala Arg Glu Asn 130 135 140Glu Met Asp Glu Asn Leu Glu Gln Val Ser Gly Ile Ile Gly Asn Leu145 150 155 160Arg His Met Ala Leu Asp Met Gly Asn Glu Ile Asp Thr Gln Asn Arg 165 170 175Gln Ile Asp Arg Ile Met Glu Lys Ala Asp Ser Asn Lys Thr Arg Ile 180 185 190Asp Glu Ala Asn Gln Arg Ala Thr Lys Met Leu Gly Ser Gly 195 200 2059206PRTRattus norvegicus 9Met Ala Glu Asp Ala Asp Met Arg Asn Glu Leu Glu Glu Met Gln Arg1 5 10 15Arg Ala Asp Gln Leu Ala Asp Glu Ser Leu Glu Ser Thr Arg Arg Met 20 25 30Leu Gln Leu Val Glu Glu Ser Lys Asp Ala Gly Ile Arg Thr Leu Val 35 40 45Met Leu Asp Glu Gln Gly Glu Gln Leu Glu Arg Ile Glu Glu Gly Met 50 55 60Asp Gln Ile Asn Lys Asp Met Lys Glu Ala Glu Lys Asn Leu Thr Asp65 70 75 80Leu Gly Lys Phe Cys Gly Leu Cys Val Cys Pro Cys Asn Lys Leu Lys 85 90 95Ser Ser Asp Ala Tyr Lys Lys Ala Trp Gly Asn Asn Gln Asp Gly Val 100 105 110Val Ala Ser Gln Pro Ala Arg Val Val Asp Glu Arg Glu Gln Met Ala 115 120 125Ile Ser Gly Gly Phe Ile Arg Arg Val Thr Asn Asp Ala Arg Glu Asn 130 135 140Glu Met Asp Glu Asn Leu Glu Gln Val Ser Gly Ile Ile Gly Asn Leu145 150 155 160Arg His Met Ala Leu Asp Met Gly Asn Glu Ile Asp Thr Gln Asn Arg 165 170 175Gln Ile Asp Arg Ile Met Glu Lys Ala Asp Ser Asn Lys Thr Arg Ile 180 185 190Asp Glu Ala Asn Gln Arg Ala Thr Lys Met Leu Gly Ser Gly 195 200 20510206PRTMus musculus 10Met Ala Glu Asp Ala Asp Met Arg Asn Glu Leu Glu Glu Met Gln Arg1 5 10 15Arg Ala Asp Gln Leu Ala Asp Glu Ser Leu Glu Ser Thr Arg Arg Met 20 25 30Leu Gln Leu Val Glu Glu Ser Lys Asp Ala Gly Ile Arg Thr Leu Val 35 40 45Met Leu Asp Glu Gln Gly Glu Gln Leu Glu Arg Ile Glu Glu Gly Met 50 55 60Asp Gln Ile Asn Lys Asp Met Lys Glu Ala Glu Lys Asn Leu Thr Asp65 70 75 80Leu Gly Lys Phe Cys Gly Leu Cys Val Cys Pro Cys Asn Lys Leu Lys 85 90 95Ser Ser Asp Ala Tyr Lys Lys Ala Trp Gly Asn Asn Gln Asp Gly Val 100 105 110Val Ala Ser Gln Pro Ala Arg Val Val Asp Glu Arg Glu Gln Met Ala 115 120 125Ile Ser Gly Gly Phe Ile Arg Arg Val Thr Asn Asp Ala Arg Glu Asn 130 135 140Glu Met Asp Glu Asn Leu Glu Gln Val Ser Gly Ile Ile Gly Asn Leu145 150 155 160Arg His Met Ala Leu Asp Met Gly Asn Glu Ile Asp Thr Gln Asn Arg 165 170 175Gln Ile Asp Arg Ile Met Glu Lys Ala Asp Ser Asn Lys Thr Arg Ile 180 185 190Asp Glu Ala Asn Gln Arg Ala Thr Lys Met Leu Gly Ser Gly 195 200 20511206PRTGallus gallus 11Met Ala Glu Asp Ala Asp Met Arg Asn Glu Leu Glu Glu Met Gln Arg1 5 10 15Arg Ala Asp Gln Leu Ala Asp Glu Ser Leu Glu Ser Thr Arg Arg Met 20 25 30Leu Gln Leu Val Glu Glu Ser Lys Asp Ala Gly Ile Arg Thr Leu Val 35 40 45Met Leu Asp Glu Gln Gly Glu Gln Leu Glu Arg Ile Glu Glu Gly Met 50 55 60Asp Gln Ile Asn Lys Asp Met Lys Glu Ala Glu Lys Asn Leu Thr Asp65 70 75 80Leu Gly Lys Phe Cys Gly Leu Cys Val Cys Pro Cys Asn Lys Leu Lys 85 90 95Ser Ser Asp Ala Tyr Lys Lys Ala Trp Gly Asn Asn Gln Asp Gly Val 100 105 110Val Ala Ser Gln Pro Ala Arg Val Val Asp Glu Arg Glu Gln Met Ala 115 120 125Ile Ser Gly Gly Phe Ile Arg Arg Val Thr Asn Asp Ala Arg Glu Asn 130 135 140Glu Met Asp Glu Asn Leu Glu Gln Val Ser Gly Ile Ile Gly Asn Leu145 150 155 160Arg His Met Ala Leu Asp Met Gly Asn Glu Ile Asp Thr Gln Asn Arg 165 170 175Gln Ile Asp Arg Ile Met Glu Lys Ala Asp Ser Asn Lys Thr Arg Ile 180 185 190Asp Glu Ala Asn Gln Arg Ala Thr Lys Met Leu Gly Ser Gly 195 200 20512204PRTCarassius auratus 12Met Ala Glu Asp Ala Asp Met Arg Asn Glu Leu Ser Asp Met Gln Gln1 5 10 15Arg Ala Asp Gln Leu Ala Asp Glu Ser Leu Glu Ser Thr Arg Arg Met 20 25 30Leu Gln Leu Val Glu Glu Ser Lys Asp Ala Gly Ile Arg Thr Leu Val 35 40 45Met Leu Asp Glu Gln Gly Glu Gln Leu Glu Arg Ile Glu Glu Gly Met 50 55 60Asp Gln Ile Asn Lys Asp Met Lys Asp Ala Glu Lys Asn Leu Asn Asp65 70 75 80Leu Gly Lys Phe Cys Gly Leu Cys Ser Cys Pro Cys Asn Lys Met Lys 85 90 95Ser Gly Gly Ser Lys Ala Trp Gly Asn Asn Gln Asp Gly Val Val Ala 100 105 110Ser Gln Pro Ala Arg Val Val Asp Glu Arg Glu Gln Met Ala Ile Ser 115 120 125Gly Gly Phe Ile Arg Arg Val Thr Asp Asp Ala Arg Glu Asn Glu Met 130 135 140Asp Glu Asn Leu Glu Gln Val Gly Gly Ile Ile Gly Asn Leu Arg His145 150 155 160Met Ala Leu Asp Met Gly Asn Glu Ile Asp Thr Gln Asn Arg Gln Ile 165 170 175Asp Arg Ile Met Glu Lys Ala Asp Ser Asn Lys Thr Arg Ile Asp Glu 180 185 190Ala Asn Gln Arg Ala Thr Lys Met Leu Gly Ser Gly 195 20013203PRTCarassius auratus 13Met Ala Asp Glu Ala Asp Met Arg Asn Glu Leu Thr Asp Met Gln Ala1 5 10 15Arg Ala Asp Gln Leu Gly Asp Glu Ser Leu Glu Ser Thr Arg Arg Met 20 25 30Leu Gln Leu Val Glu Glu Ser Lys Asp Ala Gly Ile Arg Thr Leu Val 35 40 45Met Leu Asp Glu Gln Gly Glu Gln Leu Glu Arg Ile Glu Glu Gly Met 50 55 60Asp Gln Ile Asn Lys Asp Met Lys Glu Ala Glu Lys Asn Leu Thr Asp65 70 75 80Leu Gly Asn Leu Cys Gly Leu Cys Pro Cys Pro Cys Asn Lys Leu Lys 85 90 95Gly Gly Gly Gln Ser Trp Gly Asn Asn Gln Asp Gly Val Val Ser Ser 100 105 110Gln Pro Ala Arg Val Val Asp Glu Arg Glu Gln Met Ala Ile Ser Gly 115 120 125Gly Phe Ile Arg Arg Val Thr Asn Asp Ala Arg Glu Asn Glu Met Asp 130 135 140Glu Asn Leu Glu Gln Val Gly Ser Ile Ile Gly Asn Leu Arg His Met145 150 155 160Ala Leu Asp Met Gly Asn Glu Ile Asp Thr Gln Asn Arg Gln Ile Asp 165 170 175Arg Ile Met Asp Met Ala Asp Ser Asn Lys Thr Arg Ile Asp Glu Ala 180 185 190Asn Gln Arg Ala Thr Lys Met Leu Gly Ser Gly 195 20014204PRTDanio rerio 14Met Ala Glu Asp Ser Asp Met Arg Asn Glu Leu Ala Asp Met Gln Gln1 5 10 15Arg Ala Asp Gln Leu Ala Asp Glu Ser Leu Glu Ser Thr Arg Arg Met 20 25 30Leu Gln Leu Val Glu Glu Ser Lys Asp Ala Gly Ile Arg Thr Leu Val 35 40 45Met Leu Asp Glu Gln Gly Glu Gln Leu Glu Arg Ile Glu Glu Gly Met 50 55 60Asp Gln Ile Asn Lys Asp Met Lys Asp Ala Glu Lys Asn Leu Asn Asp65 70 75 80Leu Gly Lys Phe Cys Gly Leu Cys Ser Cys Pro Cys Asn Lys Met Lys 85 90 95Ser Gly Ala Ser Lys Ala Trp Gly Asn Asn Gln Asp Gly Val Val Ala 100 105 110Ser Gln Pro Ala Arg Val Val Asp Glu Arg Glu Gln Met Ala Ile Ser 115 120 125Gly Gly Phe Ile Arg Arg Val Thr Asp Asp Ala Arg Glu Asn Glu Met 130 135 140Asp Glu Asn Leu Glu Gln Val Gly Gly Ile Ile Gly Asn Leu Arg His145 150 155 160Met Ala Leu Asp Met Gly Asn Glu Ile Asp Thr Gln Asn Arg Gln Ile 165 170 175Asp Arg Ile Met Glu Lys Ala Asp Ser Asn Lys Thr Arg Ile Asp Glu 180 185 190Ala Asn Gln Arg Ala Thr Lys Met Leu Gly Ser Gly 195 20015203PRTDanio rerio 15Met Ala Asp Glu Ser Asp Met Arg Asn Glu Leu Asn Asp Met Gln Ala1 5 10 15Arg Ala Asp Gln Leu Gly Asp Glu Ser Leu Glu Ser Thr Arg Arg Met 20 25 30Leu Gln Leu Val Glu Glu Ser Lys Asp Ala Gly Ile Arg Thr Leu Val 35 40 45Met Leu Asp Glu Gln Gly Glu Gln Leu Glu Arg Ile Glu Glu Gly Met 50 55 60Asp Gln Ile Asn Lys Asp Met Lys Glu Ala Glu Lys Asn Leu Thr Asp65 70 75 80Leu Gly Asn Leu Cys Gly Leu Cys Pro Cys Pro Cys Asn Lys Leu Lys 85 90 95Gly Gly Gly Gln Ser Trp Gly Asn Asn Gln Asp Gly Val Val Ser Ser 100 105 110Gln Pro Ala Arg Val Val Asp Glu Arg Glu Gln Met Ala Ile Ser Gly 115 120

125Gly Phe Ile Arg Arg Val Thr Asn Asp Ala Arg Glu Asn Glu Met Asp 130 135 140Glu Asn Leu Glu Gln Val Gly Ser Ile Ile Gly Asn Leu Arg His Met145 150 155 160Ala Leu Asp Met Gly Asn Glu Ile Asp Thr Gln Asn Arg Gln Ile Asp 165 170 175Arg Ile Met Asp Met Ala Asp Ser Asn Lys Thr Arg Ile Asp Glu Ala 180 185 190Asn Gln Arg Ala Thr Lys Met Leu Gly Ser Gly 195 20016210PRTTorpedo marmorata 16Met Glu Asn Ser Val Glu Asn Ser Met Asp Pro Arg Ser Glu Gln Glu1 5 10 15Glu Met Gln Arg Cys Ala Asp Gln Ile Thr Asp Glu Ser Leu Glu Ser 20 25 30Thr Arg Arg Met Leu Gln Leu Val Glu Glu Ser Lys Asp Ala Gly Ile 35 40 45Arg Thr Leu Val Met Leu Asp Glu Gln Gly Glu Gln Leu Glu Arg Ile 50 55 60Glu Glu Gly Met Asp Gln Ile Asn Lys Asp Met Lys Glu Ala Glu Lys65 70 75 80Asn Leu Ser Asp Leu Gly Lys Cys Cys Gly Leu Cys Ser Cys Pro Cys 85 90 95Asn Lys Leu Lys Asn Phe Glu Ala Gly Gly Ala Tyr Lys Lys Val Trp 100 105 110Gly Asn Asn Gln Asp Gly Val Val Ala Ser Gln Pro Ala Arg Val Met 115 120 125Asp Asp Arg Glu Gln Met Ala Met Ser Gly Gly Tyr Ile Arg Arg Ile 130 135 140Thr Asp Asp Ala Arg Glu Asn Glu Met Glu Glu Asn Leu Asp Gln Val145 150 155 160Gly Ser Ile Ile Gly Asn Leu Arg His Met Ala Leu Asp Met Ser Asn 165 170 175Glu Ile Gly Ser Gln Asn Ala Gln Ile Asp Arg Ile Val Val Lys Gly 180 185 190Asp Met Asn Lys Ala Arg Ile Asp Glu Ala Asn Lys His Ala Thr Lys 195 200 205Met Leu 21017206PRTXenopus laevis 17Met Ala Asp Asp Ala Asp Met Arg Asn Glu Leu Glu Glu Met Gln Arg1 5 10 15Arg Ala Asp Gln Leu Ala Asp Glu Ser Leu Glu Ser Thr Arg Arg Met 20 25 30Leu Gln Tyr Val Glu Gly Ser Lys Asp Ala Gly Ile Arg Thr Leu Val 35 40 45Met Leu Asp Glu Gln Gly Glu Gln Leu Asp Arg Val Glu Glu Gly Met 50 55 60Asn His Ile Asn Gln Asp Met Lys Glu Ala Glu Lys Asn Leu Lys Asp65 70 75 80Leu Gly Lys Cys Cys Gly Leu Phe Ile Cys Pro Cys Asn Lys Leu Lys 85 90 95Ser Ser Gly Ala Tyr Asn Lys Ala Trp Gly Asn Asn Gln Asp Gly Val 100 105 110Val Ala Ser Gln Pro Ala Arg Val Val Asp Glu Arg Glu Gln Met Ala 115 120 125Ile Ser Gly Gly Phe Val Arg Arg Val Thr Asn Asp Ala Arg Glu Thr 130 135 140Glu Met Asp Glu Asn Leu Glu Gln Val Gly Gly Ile Ile Gly Asn Leu145 150 155 160Arg His Met Ala Leu Asp Met Gly Asn Glu Ile Asp Thr Gln Asn Arg 165 170 175Gln Ile Asp Arg Ile Met Glu Lys Ala Asp Ser Asn Lys Ala Arg Ile 180 185 190Asp Glu Ala Asn Lys His Ala Thr Lys Met Leu Gly Ser Gly 195 200 20518206PRTXenopus laevis 18Met Ala Asp Asp Ala Asp Met Arg Asn Glu Leu Glu Glu Met Gln Arg1 5 10 15Arg Ala Asp Gln Leu Ala Asp Glu Ser Leu Glu Ser Thr Arg Arg Met 20 25 30Leu Gln Tyr Val Glu Gly Ser Lys Asp Ala Gly Ile Arg Thr Leu Val 35 40 45Met Leu Asp Glu Gln Gly Glu Gln Leu Glu Arg Ile Glu Glu Gly Met 50 55 60Glu Gln Ile Asn Lys Asp Met Lys Glu Ala Glu Lys Asn Leu Thr Asp65 70 75 80Leu Gly Lys Phe Cys Gly Leu Cys Val Cys Pro Cys Asn Lys Leu Lys 85 90 95Ser Ser Asp Ala Tyr Lys Lys Ala Trp Gly Asn Asn Gln Asp Gly Val 100 105 110Val Ala Ser Gln Pro Ala Arg Val Val Asp Glu Arg Glu Gln Met Ala 115 120 125Ile Ser Gly Gly Phe Val Arg Arg Val Thr Asn Asp Ala Arg Glu Thr 130 135 140Glu Met Asp Glu Asn Leu Glu Gln Val Gly Gly Ile Ile Gly Asn Leu145 150 155 160Arg His Met Ala Leu Asp Met Gly Asn Glu Ile Asp Thr Gln Asn Arg 165 170 175Gln Ile Asp Arg Ile Met Glu Lys Ala Asp Ser Asn Lys Ala Arg Ile 180 185 190Asp Glu Ala Asn Lys His Ala Thr Lys Met Leu Gly Ser Gly 195 200 20519212PRTStrongylocentrotus purpuratus 19Met Glu Asp Gln Asn Asp Met Asn Met Arg Ser Glu Leu Glu Glu Ile1 5 10 15Gln Met Gln Ser Asn Met Gln Thr Asp Glu Ser Leu Glu Ser Thr Arg 20 25 30Arg Met Leu Gln Met Ala Glu Glu Ser Gln Asp Met Gly Ile Lys Thr 35 40 45Leu Val Met Leu Asp Glu Gln Gly Glu Gln Leu Asp Arg Ile Glu Glu 50 55 60Gly Met Asp Gln Ile Asn Thr Asp Met Arg Glu Ala Glu Lys Asn Leu65 70 75 80Thr Gly Leu Glu Lys Cys Cys Gly Ile Cys Val Cys Pro Trp Lys Lys 85 90 95Leu Gly Asn Phe Glu Lys Gly Asp Asp Tyr Lys Lys Thr Trp Lys Gly 100 105 110Asn Asp Asp Gly Lys Val Asn Ser His Gln Pro Met Arg Met Glu Asp 115 120 125Asp Arg Asp Gly Cys Gly Gly Asn Ala Ser Met Ile Thr Arg Ile Thr 130 135 140Asn Asp Ala Arg Glu Asp Glu Met Asp Glu Asn Leu Thr Gln Val Ser145 150 155 160Ser Ile Val Gly Asn Leu Arg His Met Ala Ile Asp Met Gln Ser Glu 165 170 175Ile Gly Ala Gln Asn Ser Gln Val Gly Arg Ile Thr Ser Lys Ala Glu 180 185 190Ser Asn Glu Gly Arg Ile Asn Ser Ala Asp Lys Arg Ala Lys Asn Ile 195 200 205Leu Arg Asn Lys 21020212PRTDrosophila melanogaster 20Met Pro Ala Asp Pro Ser Glu Glu Val Ala Pro Gln Val Pro Lys Thr1 5 10 15Glu Leu Glu Glu Leu Gln Ile Asn Ala Gln Gly Val Ala Asp Glu Ser 20 25 30Leu Glu Ser Thr Arg Arg Met Leu Ala Leu Cys Glu Glu Ser Lys Glu 35 40 45Ala Gly Ile Arg Thr Leu Val Ala Leu Asp Asp Gln Gly Glu Gln Leu 50 55 60Asp Arg Ile Glu Glu Gly Met Asp Gln Ile Asn Ala Asp Met Arg Glu65 70 75 80Ala Glu Lys Asn Leu Ser Gly Met Glu Lys Cys Cys Gly Ile Cys Val 85 90 95Leu Pro Cys Asn Lys Ser Gln Ser Phe Lys Glu Asp Asp Gly Thr Trp 100 105 110Lys Gly Asn Asp Asp Gly Lys Val Val Asn Asn Gln Pro Gln Arg Val 115 120 125Met Asp Asp Arg Asn Gly Met Met Ala Gln Ala Gly Tyr Ile Gly Arg 130 135 140Ile Thr Asn Asp Ala Arg Glu Asp Glu Met Glu Glu Asn Met Gly Gln145 150 155 160Val Asn Thr Met Ile Gly Asn Leu Arg Asn Met Ala Leu Asp Met Gly 165 170 175Ser Glu Leu Glu Asn Gln Asn Arg Gln Ile Asp Arg Ile Asn Arg Lys 180 185 190Gly Glu Ser Asn Glu Ala Arg Ile Ala Val Ala Asn Gln Arg Ala His 195 200 205Gln Leu Leu Lys 21021212PRTHirudo medicinalis 21Met Ala Lys Asp Ile Lys Pro Lys Pro Ala Asn Gly Arg Asp Ser Pro1 5 10 15Thr Asp Leu Gln Glu Ile Gln Leu Gln Met Asn Ala Ile Thr Asp Asp 20 25 30Ser Leu Glu Ser Thr Arg Arg Met Leu Ala Met Cys Glu Glu Ser Lys 35 40 45Asp Ala Gly Ile Arg Thr Leu Val Met Leu Asp Glu Gln Gly Glu Gln 50 55 60Leu Asp Arg Ile Glu Glu Gly Met Asp Gln Ile Asn Gln Asp Met Arg65 70 75 80Asp Ala Glu Lys Asn Leu Glu Gly Met Glu Lys Cys Cys Gly Leu Cys 85 90 95Ile Leu Pro Trp Lys Arg Thr Lys Asn Phe Asp Lys Gly Ala Glu Trp 100 105 110Asn Lys Gly Asp Glu Gly Lys Val Asn Thr Asp Gly Pro Arg Leu Val 115 120 125Val Gly Asp Gly Asn Met Gly Pro Ser Gly Gly Phe Ile Thr Lys Ile 130 135 140Thr Asn Asp Ala Arg Glu Glu Glu Met Glu Gln Asn Met Gly Glu Val145 150 155 160Ser Asn Met Ile Ser Asn Leu Arg Asn Met Ala Val Asp Met Gly Ser 165 170 175Glu Ile Asp Ser Gln Asn Arg Gln Val Asp Arg Ile Asn Asn Lys Met 180 185 190Thr Ser Asn Gln Leu Arg Ile Ser Asp Ala Asn Lys Arg Ala Ser Lys 195 200 205Leu Leu Lys Glu 21022212PRTLoligo pealei 22Met Ser Ala Asn Gly Glu Val Glu Val Pro Lys Thr Glu Leu Glu Glu1 5 10 15Ile Gln Gln Gln Cys Asn Gln Val Thr Asp Asp Ser Leu Glu Ser Thr 20 25 30Arg Arg Met Leu Asn Met Cys Glu Glu Ser Lys Glu Ala Gly Ile Arg 35 40 45Thr Leu Val Met Leu Asp Glu Gln Gly Glu Gln Leu Asp Arg Ile Glu 50 55 60Glu Gly Leu Asp Gln Ile Asn Gln Asp Met Lys Asp Ala Glu Lys Asn65 70 75 80Leu Glu Gly Met Glu Lys Cys Cys Gly Leu Cys Val Leu Pro Trp Lys 85 90 95Arg Gly Lys Ser Phe Glu Lys Ser Gly Asp Tyr Ala Asn Thr Trp Lys 100 105 110Lys Asp Asp Asp Gly Pro Thr Asn Thr Asn Gly Pro Arg Val Thr Val 115 120 125Gly Asp Gln Asn Gly Met Gly Pro Ser Ser Gly Tyr Val Thr Arg Ile 130 135 140Thr Asn Asp Ala Arg Glu Asp Asp Met Glu Asn Asn Met Lys Glu Val145 150 155 160Ser Ser Met Ile Gly Asn Leu Arg Asn Met Ala Ile Asp Met Gly Asn 165 170 175Glu Ile Gly Ser Gln Asn Arg Gln Val Asp Arg Ile Gln Gln Lys Ala 180 185 190Glu Ser Asn Glu Ser Arg Ile Asp Glu Ala Asn Lys Lys Ala Thr Lys 195 200 205Leu Leu Lys Asn 21023220PRTLymnaea stagnalis 23Met Thr Thr Asn Gly Glu Ile Leu Pro Val Gly Glu Glu Glu Glu Glu1 5 10 15Glu Leu Gly Glu Asp Ala Leu Leu Arg Lys Gln Ile Asp Cys Asn Thr 20 25 30Asn Glu Ser Leu Glu Ser Thr Arg Arg Met Leu Ser Leu Cys Glu Glu 35 40 45Ser Lys Glu Ala Gly Ile Lys Thr Leu Val Met Leu Asp Glu Gln Gly 50 55 60Glu Gln Leu Asp Arg Ile Glu Glu Gly Met Gly Gln Ile Asn Gln Asp65 70 75 80Met Arg Asp Ala Glu Lys Asn Leu Glu Gly Leu Glu Lys Cys Cys Gly 85 90 95Leu Cys Val Leu Pro Trp Lys Arg Ser Lys Asn Phe Glu Lys Gly Ser 100 105 110Asp Tyr Asn Lys Thr Trp Lys Ala Ser Glu Asp Gly Lys Ile Asn Thr 115 120 125Asn Gly Pro Arg Leu Val Val Asp Gln Gly Asn Gly Ser Gly Pro Thr 130 135 140Gly Gly Tyr Ile Thr Arg Ile Thr Asn Asp Ala Arg Glu Asp Glu Met145 150 155 160Glu Gln Asn Ile Gly Glu Val Ala Gly Met Val Ser Asn Leu Arg Asn 165 170 175Met Ala Val Asp Met Gly Asn Glu Ile Glu Ser Gln Asn Lys Gln Leu 180 185 190Asp Arg Ile Asn Gln Lys Gly Gly Ser Leu Asn Val Arg Val Asp Glu 195 200 205Ala Asn Lys Arg Ala Asn Arg Ile Leu Arg Lys Gln 210 215 22024207PRTCaenorhabditis elegans 24Met Ser Gly Asp Asp Asp Ile Pro Glu Gly Leu Glu Ala Ile Asn Leu1 5 10 15Lys Met Asn Ala Thr Thr Asp Asp Ser Leu Glu Ser Thr Arg Arg Met 20 25 30Leu Ala Leu Cys Glu Glu Ser Lys Glu Ala Gly Ile Lys Thr Leu Val 35 40 45Met Leu Asp Asp Gln Gly Glu Gln Leu Glu Arg Cys Glu Gly Ala Leu 50 55 60Asp Thr Ile Asn Gln Asp Met Lys Glu Ala Glu Asp His Leu Lys Gly65 70 75 80Met Glu Lys Cys Cys Gly Leu Cys Val Leu Pro Trp Asn Lys Thr Asp 85 90 95Asp Phe Glu Lys Thr Glu Phe Ala Lys Ala Trp Lys Lys Asp Asp Asp 100 105 110Gly Gly Val Ile Ser Asp Gln Pro Arg Ile Thr Val Gly Asp Ser Ser 115 120 125Met Gly Pro Gln Gly Gly Tyr Ile Thr Lys Ile Thr Asn Asp Ala Arg 130 135 140Glu Asp Glu Met Asp Glu Asn Val Gln Gln Val Ser Thr Met Val Gly145 150 155 160Asn Leu Arg Asn Met Ala Ile Asp Met Ser Thr Glu Val Ser Asn Gln 165 170 175Asn Arg Gln Leu Asp Arg Ile His Asp Lys Ala Gln Ser Asn Glu Val 180 185 190Arg Val Glu Ser Ala Asn Lys Arg Ala Lys Asn Leu Ile Thr Lys 195 200 20525808PRTHomo sapiens 25Met Gly Ala Pro Ala Cys Ala Leu Ala Leu Cys Val Ala Val Ala Ile1 5 10 15Val Ala Gly Ala Ser Ser Glu Ser Leu Gly Thr Glu Gln Arg Val Val 20 25 30Gly Arg Ala Ala Glu Val Pro Gly Pro Glu Pro Gly Gln Gln Glu Gln 35 40 45Leu Val Phe Gly Ser Gly Asp Ala Val Glu Leu Ser Cys Pro Pro Pro 50 55 60Gly Gly Gly Pro Met Gly Pro Thr Val Trp Val Lys Asp Gly Thr Gly65 70 75 80Leu Val Pro Ser Glu Arg Val Leu Val Gly Pro Gln Arg Leu Gln Val 85 90 95Leu Asn Ala Ser His Glu Asp Ser Gly Ala Tyr Ser Cys Arg Gln Arg 100 105 110Leu Thr Gln Arg Val Leu Cys His Phe Ser Val Arg Val Thr Asp Ala 115 120 125Pro Ser Ser Gly Asp Asp Glu Asp Gly Glu Asp Glu Ala Glu Asp Thr 130 135 140Gly Val Asp Thr Gly Ala Pro Tyr Trp Thr Arg Pro Glu Arg Met Asp145 150 155 160Lys Lys Leu Leu Ala Val Pro Ala Ala Asn Thr Val Arg Phe Arg Cys 165 170 175Pro Ala Ala Gly Asn Pro Thr Pro Ser Ile Ser Trp Leu Lys Asn Gly 180 185 190Arg Glu Phe Arg Gly Glu His Arg Ile Gly Gly Ile Lys Leu Arg His 195 200 205Gln Gln Trp Ser Leu Val Met Glu Ser Val Val Pro Ser Asp Arg Gly 210 215 220Asn Tyr Thr Cys Val Val Glu Asn Lys Phe Gly Ser Ile Arg Gln Thr225 230 235 240Tyr Thr Leu Asp Val Leu Glu Arg Ser Pro His Arg Pro Ile Leu Gln 245 250 255Ala Gly Leu Pro Ala Asn Gln Thr Ala Val Leu Gly Ser Asp Val Glu 260 265 270Phe His Cys Lys Val Tyr Ser Asp Ala Gln Pro His Ile Gln Trp Leu 275 280 285Lys His Val Glu Val Asn Gly Ser Lys Val Gly Pro Asp Gly Thr Pro 290 295 300Tyr Val Thr Val Leu Lys Ser Trp Ile Ser Glu Ser Val Glu Ala Asp305 310 315 320Val Arg Leu Arg Leu Ala Asn Val Ser Glu Arg Asp Gly Gly Glu Tyr 325 330 335Leu Cys Arg Ala Thr Asn Phe Ile Gly Val Ala Glu Lys Ala Phe Trp 340 345 350Leu Ser Val His Gly Pro Arg Ala Ala Glu Glu Glu Leu Val Glu Ala 355 360 365Asp Glu Ala Gly Ser Val Tyr Ala Gly Ile Leu Ser Tyr Gly Val Gly 370 375 380Phe Phe Leu Phe Ile Leu Val Val Ala Ala Val Thr Leu Cys Arg Leu385 390 395 400Arg Ser Pro Pro Lys Lys Gly Leu Gly Ser Pro Thr Val His Lys Ile 405 410 415Ser Arg Phe Pro Leu Lys Arg Gln Val Ser Leu Glu Ser Asn Ala Ser 420 425 430Met Ser Ser Asn Thr Pro Leu Val Arg Ile Ala Arg Leu Ser Ser Gly 435 440 445Glu Gly Pro Thr Leu Ala Asn Val Ser Glu Leu Glu Leu Pro Ala Asp 450 455 460Pro Lys Trp Glu Leu Ser Arg Ala

Arg Leu Thr Leu Gly Lys Pro Leu465 470 475 480Gly Glu Gly Cys Phe Gly Gln Val Val Met Ala Glu Ala Ile Gly Ile 485 490 495Asp Lys Asp Arg Ala Ala Lys Pro Val Thr Val Ala Val Lys Met Leu 500 505 510Lys Asp Asp Ala Thr Asp Lys Asp Leu Ser Asp Leu Val Ser Glu Met 515 520 525Glu Met Met Lys Met Ile Gly Lys His Lys Asn Ile Ile Asn Leu Leu 530 535 540Gly Ala Cys Thr Gln Gly Gly Pro Leu Tyr Val Leu Val Glu Tyr Ala545 550 555 560Ala Lys Gly Asn Leu Arg Glu Phe Leu Arg Ala Arg Arg Pro Pro Gly 565 570 575Leu Asp Tyr Ser Phe Asp Thr Cys Lys Pro Pro Glu Glu Gln Leu Thr 580 585 590Phe Lys Asp Leu Val Ser Cys Ala Tyr Gln Val Ala Arg Gly Met Glu 595 600 605Tyr Leu Ala Ser Gln Lys Cys Ile His Arg Asp Leu Ala Ala Arg Asn 610 615 620Val Leu Val Thr Glu Asp Asn Val Met Lys Ile Ala Asp Phe Gly Leu625 630 635 640Ala Arg Asp Val His Asn Leu Asp Tyr Tyr Lys Lys Thr Thr Asn Gly 645 650 655Arg Leu Pro Val Lys Trp Met Ala Pro Glu Ala Leu Phe Asp Arg Val 660 665 670Tyr Thr His Gln Ser Asp Val Trp Ser Phe Gly Val Leu Leu Trp Glu 675 680 685Ile Phe Thr Leu Gly Gly Ser Pro Tyr Pro Gly Ile Pro Val Glu Glu 690 695 700Leu Phe Lys Leu Leu Lys Glu Gly His Arg Met Asp Lys Pro Ala Asn705 710 715 720Cys Thr His Asp Leu Tyr Met Ile Met Arg Glu Cys Trp His Ala Ala 725 730 735Pro Ser Gln Arg Pro Thr Phe Lys Gln Leu Val Glu Asp Leu Asp Arg 740 745 750Val Leu Thr Val Thr Ser Thr Asp Glu Tyr Leu Asp Leu Ser Ala Pro 755 760 765Phe Glu Gln Tyr Ser Pro Gly Gly Gln Asp Thr Pro Ser Ser Ser Ser 770 775 780Ser Gly Asp Asp Ser Val Phe Ala His Asp Leu Leu Pro Pro Ala Pro785 790 795 800Pro Ser Ser Gly Gly Ser Arg Thr 80526806PRTHomo sapiens 26Met Gly Ala Pro Ala Cys Ala Leu Ala Leu Cys Val Ala Val Ala Ile1 5 10 15Val Ala Gly Ala Ser Ser Glu Ser Leu Gly Thr Glu Gln Arg Val Val 20 25 30Gly Arg Ala Ala Glu Val Pro Gly Pro Glu Pro Gly Gln Gln Glu Gln 35 40 45Leu Val Phe Gly Ser Gly Asp Ala Val Glu Leu Ser Cys Pro Pro Pro 50 55 60Gly Gly Gly Pro Met Gly Pro Thr Val Trp Val Lys Asp Gly Thr Gly65 70 75 80Leu Val Pro Ser Glu Arg Val Leu Val Gly Pro Gln Arg Leu Gln Val 85 90 95Leu Asn Ala Ser His Glu Asp Ser Gly Ala Tyr Ser Cys Arg Gln Arg 100 105 110Leu Thr Gln Arg Val Leu Cys His Phe Ser Val Arg Val Thr Asp Ala 115 120 125Pro Ser Ser Gly Asp Asp Glu Asp Gly Glu Asp Glu Ala Glu Asp Thr 130 135 140Gly Val Asp Thr Gly Ala Pro Tyr Trp Thr Arg Pro Glu Arg Met Asp145 150 155 160Lys Lys Leu Leu Ala Val Pro Ala Ala Asn Thr Val Arg Phe Arg Cys 165 170 175Pro Ala Ala Gly Asn Pro Thr Pro Ser Ile Ser Trp Leu Lys Asn Gly 180 185 190Arg Glu Phe Arg Gly Glu His Arg Ile Gly Gly Ile Lys Leu Arg His 195 200 205Gln Gln Trp Ser Leu Val Met Glu Ser Val Val Pro Ser Asp Arg Gly 210 215 220Asn Tyr Thr Cys Val Val Glu Asn Lys Phe Gly Ser Ile Arg Gln Thr225 230 235 240Tyr Thr Leu Asp Val Leu Glu Arg Ser Pro His Arg Pro Ile Leu Gln 245 250 255Ala Gly Leu Pro Ala Asn Gln Thr Ala Val Leu Gly Ser Asp Val Glu 260 265 270Phe His Cys Lys Val Tyr Ser Asp Ala Gln Pro His Ile Gln Trp Leu 275 280 285Lys His Val Glu Val Asn Gly Ser Lys Val Gly Pro Asp Gly Thr Pro 290 295 300Tyr Val Thr Val Leu Lys Thr Ala Gly Ala Asn Thr Thr Asp Lys Glu305 310 315 320Leu Glu Val Leu Ser Leu His Asn Val Thr Phe Glu Asp Ala Gly Glu 325 330 335Tyr Thr Cys Leu Ala Gly Asn Ser Ile Gly Phe Ser His His Ser Ala 340 345 350Trp Leu Val Val Leu Pro Ala Glu Glu Glu Leu Val Glu Ala Asp Glu 355 360 365Ala Gly Ser Val Tyr Ala Gly Ile Leu Ser Tyr Gly Val Gly Phe Phe 370 375 380Leu Phe Ile Leu Val Val Ala Ala Val Thr Leu Cys Arg Leu Arg Ser385 390 395 400Pro Pro Lys Lys Gly Leu Gly Ser Pro Thr Val His Lys Ile Ser Arg 405 410 415Phe Pro Leu Lys Arg Gln Val Ser Leu Glu Ser Asn Ala Ser Met Ser 420 425 430Ser Asn Thr Pro Leu Val Arg Ile Ala Arg Leu Ser Ser Gly Glu Gly 435 440 445Pro Thr Leu Ala Asn Val Ser Glu Leu Glu Leu Pro Ala Asp Pro Lys 450 455 460Trp Glu Leu Ser Arg Ala Arg Leu Thr Leu Gly Lys Pro Leu Gly Glu465 470 475 480Gly Cys Phe Gly Gln Val Val Met Ala Glu Ala Ile Gly Ile Asp Lys 485 490 495Asp Arg Ala Ala Lys Pro Val Thr Val Ala Val Lys Met Leu Lys Asp 500 505 510Asp Ala Thr Asp Lys Asp Leu Ser Asp Leu Val Ser Glu Met Glu Met 515 520 525Met Lys Met Ile Gly Lys His Lys Asn Ile Ile Asn Leu Leu Gly Ala 530 535 540Cys Thr Gln Gly Gly Pro Leu Tyr Val Leu Val Glu Tyr Ala Ala Lys545 550 555 560Gly Asn Leu Arg Glu Phe Leu Arg Ala Arg Arg Pro Pro Gly Leu Asp 565 570 575Tyr Ser Phe Asp Thr Cys Lys Pro Pro Glu Glu Gln Leu Thr Phe Lys 580 585 590Asp Leu Val Ser Cys Ala Tyr Gln Val Ala Arg Gly Met Glu Tyr Leu 595 600 605Ala Ser Gln Lys Cys Ile His Arg Asp Leu Ala Ala Arg Asn Val Leu 610 615 620Val Thr Glu Asp Asn Val Met Lys Ile Ala Asp Phe Gly Leu Ala Arg625 630 635 640Asp Val His Asn Leu Asp Tyr Tyr Lys Lys Thr Thr Asn Gly Arg Leu 645 650 655Pro Val Lys Trp Met Ala Pro Glu Ala Leu Phe Asp Arg Val Tyr Thr 660 665 670His Gln Ser Asp Val Trp Ser Phe Gly Val Leu Leu Trp Glu Ile Phe 675 680 685Thr Leu Gly Gly Ser Pro Tyr Pro Gly Ile Pro Val Glu Glu Leu Phe 690 695 700Lys Leu Leu Lys Glu Gly His Arg Met Asp Lys Pro Ala Asn Cys Thr705 710 715 720His Asp Leu Tyr Met Ile Met Arg Glu Cys Trp His Ala Ala Pro Ser 725 730 735Gln Arg Pro Thr Phe Lys Gln Leu Val Glu Asp Leu Asp Arg Val Leu 740 745 750Thr Val Thr Ser Thr Asp Glu Tyr Leu Asp Leu Ser Ala Pro Phe Glu 755 760 765Gln Tyr Ser Pro Gly Gly Gln Asp Thr Pro Ser Ser Ser Ser Ser Gly 770 775 780Asp Asp Ser Val Phe Ala His Asp Leu Leu Pro Pro Ala Pro Pro Ser785 790 795 800Ser Gly Gly Ser Arg Thr 80527694PRTHomo sapiens 27Met Gly Ala Pro Ala Cys Ala Leu Ala Leu Cys Val Ala Val Ala Ile1 5 10 15Val Ala Gly Ala Ser Ser Glu Ser Leu Gly Thr Glu Gln Arg Val Val 20 25 30Gly Arg Ala Ala Glu Val Pro Gly Pro Glu Pro Gly Gln Gln Glu Gln 35 40 45Leu Val Phe Gly Ser Gly Asp Ala Val Glu Leu Ser Cys Pro Pro Pro 50 55 60Gly Gly Gly Pro Met Gly Pro Thr Val Trp Val Lys Asp Gly Thr Gly65 70 75 80Leu Val Pro Ser Glu Arg Val Leu Val Gly Pro Gln Arg Leu Gln Val 85 90 95Leu Asn Ala Ser His Glu Asp Ser Gly Ala Tyr Ser Cys Arg Gln Arg 100 105 110Leu Thr Gln Arg Val Leu Cys His Phe Ser Val Arg Val Thr Asp Ala 115 120 125Pro Ser Ser Gly Asp Asp Glu Asp Gly Glu Asp Glu Ala Glu Asp Thr 130 135 140Gly Val Asp Thr Gly Ala Pro Tyr Trp Thr Arg Pro Glu Arg Met Asp145 150 155 160Lys Lys Leu Leu Ala Val Pro Ala Ala Asn Thr Val Arg Phe Arg Cys 165 170 175Pro Ala Ala Gly Asn Pro Thr Pro Ser Ile Ser Trp Leu Lys Asn Gly 180 185 190Arg Glu Phe Arg Gly Glu His Arg Ile Gly Gly Ile Lys Leu Arg His 195 200 205Gln Gln Trp Ser Leu Val Met Glu Ser Val Val Pro Ser Asp Arg Gly 210 215 220Asn Tyr Thr Cys Val Val Glu Asn Lys Phe Gly Ser Ile Arg Gln Thr225 230 235 240Tyr Thr Leu Asp Val Leu Glu Arg Ser Pro His Arg Pro Ile Leu Gln 245 250 255Ala Gly Leu Pro Ala Asn Gln Thr Ala Val Leu Gly Ser Asp Val Glu 260 265 270Phe His Cys Lys Val Tyr Ser Asp Ala Gln Pro His Ile Gln Trp Leu 275 280 285Lys His Val Glu Val Asn Gly Ser Lys Val Gly Pro Asp Gly Thr Pro 290 295 300Tyr Val Thr Val Leu Lys Val Ser Leu Glu Ser Asn Ala Ser Met Ser305 310 315 320Ser Asn Thr Pro Leu Val Arg Ile Ala Arg Leu Ser Ser Gly Glu Gly 325 330 335Pro Thr Leu Ala Asn Val Ser Glu Leu Glu Leu Pro Ala Asp Pro Lys 340 345 350Trp Glu Leu Ser Arg Ala Arg Leu Thr Leu Gly Lys Pro Leu Gly Glu 355 360 365Gly Cys Phe Gly Gln Val Val Met Ala Glu Ala Ile Gly Ile Asp Lys 370 375 380Asp Arg Ala Ala Lys Pro Val Thr Val Ala Val Lys Met Leu Lys Asp385 390 395 400Asp Ala Thr Asp Lys Asp Leu Ser Asp Leu Val Ser Glu Met Glu Met 405 410 415Met Lys Met Ile Gly Lys His Lys Asn Ile Ile Asn Leu Leu Gly Ala 420 425 430Cys Thr Gln Gly Gly Pro Leu Tyr Val Leu Val Glu Tyr Ala Ala Lys 435 440 445Gly Asn Leu Arg Glu Phe Leu Arg Ala Arg Arg Pro Pro Gly Leu Asp 450 455 460Tyr Ser Phe Asp Thr Cys Lys Pro Pro Glu Glu Gln Leu Thr Phe Lys465 470 475 480Asp Leu Val Ser Cys Ala Tyr Gln Val Ala Arg Gly Met Glu Tyr Leu 485 490 495Ala Ser Gln Lys Cys Ile His Arg Asp Leu Ala Ala Arg Asn Val Leu 500 505 510Val Thr Glu Asp Asn Val Met Lys Ile Ala Asp Phe Gly Leu Ala Arg 515 520 525Asp Val His Asn Leu Asp Tyr Tyr Lys Lys Thr Thr Asn Gly Arg Leu 530 535 540Pro Val Lys Trp Met Ala Pro Glu Ala Leu Phe Asp Arg Val Tyr Thr545 550 555 560His Gln Ser Asp Val Trp Ser Phe Gly Val Leu Leu Trp Glu Ile Phe 565 570 575Thr Leu Gly Gly Ser Pro Tyr Pro Gly Ile Pro Val Glu Glu Leu Phe 580 585 590Lys Leu Leu Lys Glu Gly His Arg Met Asp Lys Pro Ala Asn Cys Thr 595 600 605His Asp Leu Tyr Met Ile Met Arg Glu Cys Trp His Ala Ala Pro Ser 610 615 620Gln Arg Pro Thr Phe Lys Gln Leu Val Glu Asp Leu Asp Arg Val Leu625 630 635 640Thr Val Thr Ser Thr Asp Glu Tyr Leu Asp Leu Ser Ala Pro Phe Glu 645 650 655Gln Tyr Ser Pro Gly Gly Gln Asp Thr Pro Ser Ser Ser Ser Ser Gly 660 665 670Asp Asp Ser Val Phe Ala His Asp Leu Leu Pro Pro Ala Pro Pro Ser 675 680 685Ser Gly Gly Ser Arg Thr 69028604PRTHomo sapiens 28Ala Gln Arg Arg Lys Glu Arg Glu Glu Leu Ala Gln Gln Tyr Glu Ala1 5 10 15Ile Leu Arg Glu Cys Gly His Gly Arg Phe Gln Trp Thr Leu Tyr Phe 20 25 30Val Leu Gly Leu Ala Leu Met Ala Asp Gly Val Glu Val Phe Val Val 35 40 45Gly Phe Val Leu Pro Ser Ala Glu Lys Asp Met Cys Leu Ser Asp Ser 50 55 60Asn Lys Gly Met Leu Gly Leu Ile Val Tyr Leu Gly Met Met Val Gly65 70 75 80Ala Phe Leu Trp Gly Gly Leu Ala Asp Arg Leu Gly Arg Arg Gln Cys 85 90 95Leu Leu Ile Ser Leu Ser Val Asn Ser Val Phe Ala Phe Phe Ser Ser 100 105 110Phe Val Gln Gly Tyr Gly Thr Phe Leu Phe Cys Arg Leu Leu Ser Gly 115 120 125Val Gly Ile Gly Gly Ser Ile Pro Ile Val Phe Ser Tyr Phe Ser Glu 130 135 140Phe Leu Ala Gln Glu Lys Arg Gly Glu His Leu Ser Trp Leu Cys Met145 150 155 160Phe Trp Met Ile Gly Gly Val Tyr Ala Ala Ala Met Ala Trp Ala Ile 165 170 175Ile Pro His Tyr Gly Trp Ser Phe Gln Met Gly Ser Ala Tyr Gln Phe 180 185 190His Ser Trp Arg Val Phe Val Leu Val Cys Ala Phe Pro Ser Val Phe 195 200 205Ala Ile Gly Ala Leu Thr Thr Gln Pro Glu Ser Pro Arg Phe Phe Leu 210 215 220Glu Asn Gly Lys His Asp Glu Ala Trp Met Val Leu Lys Gln Val His225 230 235 240Asp Thr Asn Met Arg Ala Lys Gly His Pro Glu Arg Val Phe Ser Val 245 250 255Thr His Ile Lys Thr Ile His Gln Glu Asp Glu Leu Ile Glu Ile Gln 260 265 270Ser Asp Thr Gly Thr Trp Tyr Gln Arg Trp Gly Val Arg Ala Leu Ser 275 280 285Leu Gly Gly Gln Val Trp Gly Asn Phe Leu Ser Cys Phe Gly Pro Glu 290 295 300Tyr Arg Arg Ile Thr Leu Met Met Met Gly Val Trp Phe Thr Met Ser305 310 315 320Phe Ser Tyr Tyr Gly Leu Thr Val Trp Phe Pro Asp Met Ile Arg His 325 330 335Leu Gln Ala Val Asp Tyr Ala Ser Arg Thr Lys Val Phe Pro Gly Glu 340 345 350Arg Val Glu His Val Thr Phe Asn Phe Thr Leu Glu Asn Gln Ile His 355 360 365Arg Gly Gly Gln Tyr Phe Asn Asp Lys Phe Ile Gly Leu Arg Leu Lys 370 375 380Ser Val Ser Phe Glu Asp Ser Leu Phe Glu Glu Cys Tyr Phe Glu Asp385 390 395 400Val Thr Ser Ser Asn Thr Phe Phe Arg Asn Cys Thr Phe Ile Asn Thr 405 410 415Val Phe Tyr Asn Thr Asp Leu Phe Glu Tyr Lys Phe Val Asn Ser Arg 420 425 430Leu Ile Asn Ser Thr Phe Leu His Asn Lys Glu Gly Cys Pro Leu Asp 435 440 445Val Thr Gly Thr Gly Glu Gly Ala Tyr Met Val Tyr Phe Val Ser Phe 450 455 460Leu Gly Thr Leu Ala Val Leu Pro Gly Asn Ile Val Ser Ala Leu Leu465 470 475 480Met Asp Lys Ile Gly Arg Leu Arg Met Leu Ala Gly Ser Ser Val Met 485 490 495Ser Cys Val Ser Cys Phe Phe Leu Ser Phe Gly Asn Ser Glu Ser Ala 500 505 510Met Ile Ala Leu Leu Cys Leu Phe Gly Gly Val Ser Ile Ala Ser Trp 515 520 525Asn Ala Leu Asp Val Leu Thr Val Glu Leu Tyr Pro Ser Asp Lys Arg 530 535 540Thr Thr Ala Phe Gly Phe Leu Asn Ala Leu Cys Lys Leu Ala Ala Val545 550 555 560Leu Gly Ile Ser Ile Phe Thr Ser Phe Val Gly Ile Thr Lys Ala Ala 565 570 575Pro Ile Leu Phe Ala Ser Ala Ala Leu Ala Leu Gly Ser Ser Leu Ala 580 585 590Leu Lys Leu Pro Glu Thr Arg Gly Gln Val Leu Gln 595 60029683PRTHomo sapiens 29Met Asp Asp Tyr Lys Tyr Gln Asp Asn Tyr Gly Gly Tyr Ala Pro Ser1 5 10 15Asp Gly Tyr Tyr Arg Gly Asn Glu Ser

Asn Pro Glu Glu Asp Ala Gln 20 25 30Ser Asp Val Thr Glu Gly His Asp Glu Glu Asp Glu Ile Tyr Glu Gly 35 40 45Glu Tyr Gln Gly Ile Pro His Pro Asp Asp Val Lys Ala Lys Gln Ala 50 55 60Lys Met Ala Pro Ser Arg Met Asp Ser Leu Arg Gly Gln Thr Asp Leu65 70 75 80Met Ala Glu Arg Leu Glu Asp Glu Glu Gln Leu Ala His Gln Tyr Glu 85 90 95Thr Ile Met Asp Glu Cys Gly His Gly Arg Phe Gln Trp Ile Leu Phe 100 105 110Phe Val Leu Gly Leu Ala Leu Met Ala Asp Gly Val Glu Val Phe Val 115 120 125Val Ser Phe Ala Leu Pro Ser Ala Glu Lys Asp Met Cys Leu Ser Ser 130 135 140Ser Lys Lys Gly Met Leu Gly Met Ile Val Tyr Leu Gly Met Met Ala145 150 155 160Gly Ala Phe Ile Leu Gly Gly Leu Ala Asp Lys Leu Gly Arg Lys Arg 165 170 175Val Leu Ser Met Ser Leu Ala Val Asn Ala Ser Phe Ala Ser Leu Ser 180 185 190Ser Phe Val Gln Gly Tyr Gly Ala Phe Leu Phe Cys Arg Leu Ile Ser 195 200 205Gly Ile Gly Ile Gly Gly Ala Leu Pro Ile Val Phe Ala Tyr Phe Ser 210 215 220Glu Phe Leu Ser Arg Glu Lys Arg Gly Glu His Leu Ser Trp Leu Gly225 230 235 240Ile Phe Trp Met Thr Gly Gly Leu Tyr Ala Ser Ala Met Ala Trp Ser 245 250 255Ile Ile Pro His Tyr Gly Trp Gly Phe Ser Met Gly Thr Asn Tyr His 260 265 270Phe His Ser Trp Arg Val Phe Val Ile Val Cys Ala Leu Pro Cys Thr 275 280 285Val Ser Met Val Ala Leu Lys Phe Met Pro Glu Ser Pro Arg Phe Leu 290 295 300Leu Glu Met Gly Lys His Asp Glu Ala Trp Met Ile Leu Lys Gln Val305 310 315 320His Asp Thr Asn Met Arg Ala Lys Gly Thr Pro Glu Lys Val Phe Thr 325 330 335Val Ser Asn Ile Lys Thr Pro Lys Gln Met Asp Glu Phe Ile Glu Ile 340 345 350Gln Ser Ser Thr Gly Thr Trp Tyr Gln Arg Trp Leu Val Arg Phe Lys 355 360 365Thr Ile Phe Lys Gln Val Trp Asp Asn Ala Leu Tyr Cys Val Met Gly 370 375 380Pro Tyr Arg Met Asn Thr Leu Ile Leu Ala Val Val Trp Phe Ala Met385 390 395 400Ala Phe Ser Tyr Tyr Gly Leu Thr Val Trp Phe Pro Asp Met Ile Arg 405 410 415Tyr Phe Gln Asp Glu Glu Tyr Lys Ser Lys Met Lys Val Phe Phe Gly 420 425 430Glu His Val Tyr Gly Ala Thr Ile Asn Phe Thr Met Glu Asn Gln Ile 435 440 445His Gln His Gly Lys Leu Val Asn Asp Lys Phe Thr Arg Met Tyr Phe 450 455 460Lys His Val Leu Phe Glu Asp Thr Phe Phe Asp Glu Cys Tyr Phe Glu465 470 475 480Asp Val Thr Ser Thr Asp Thr Tyr Phe Lys Asn Cys Thr Ile Glu Ser 485 490 495Thr Ile Phe Tyr Asn Thr Asp Leu Tyr Glu His Lys Phe Ile Asn Cys 500 505 510Arg Phe Ile Asn Ser Thr Phe Leu Glu Gln Lys Glu Gly Cys His Met 515 520 525Asp Leu Glu Gln Asp Asn Asp Phe Leu Ile Tyr Leu Val Ser Phe Leu 530 535 540Gly Ser Leu Ser Val Leu Pro Gly Asn Ile Ile Ser Ala Leu Leu Met545 550 555 560Asp Arg Ile Gly Arg Leu Lys Met Ile Gly Gly Ser Met Leu Ile Ser 565 570 575Ala Val Cys Cys Phe Phe Leu Phe Phe Gly Asn Ser Glu Ser Ala Met 580 585 590Ile Gly Trp Gln Cys Leu Phe Cys Gly Thr Ser Ile Ala Ala Trp Asn 595 600 605Ala Leu Asp Val Ile Thr Val Glu Leu Tyr Pro Thr Asn Gln Arg Ala 610 615 620Thr Ala Phe Gly Ile Leu Asn Gly Leu Cys Lys Phe Gly Ala Ile Leu625 630 635 640Gly Asn Thr Ile Phe Ala Ser Phe Val Gly Ile Thr Lys Val Val Pro 645 650 655Ile Leu Leu Ala Ala Ala Ser Leu Val Gly Gly Gly Leu Ile Ala Leu 660 665 670Arg Leu Pro Glu Thr Arg Glu Gln Val Leu Ile 675 68030727PRTHomo sapiens 30Met Glu Asp Ser Tyr Lys Asp Arg Thr Ser Leu Met Lys Gly Ala Lys1 5 10 15Asp Ile Ala Arg Glu Val Lys Lys Gln Thr Val Lys Lys Val Asn Gln 20 25 30Ala Val Asp Arg Ala Gln Asp Glu Tyr Thr Gln Arg Ser Tyr Ser Arg 35 40 45Phe Gln Asp Glu Glu Asp Asp Asp Asp Tyr Tyr Pro Ala Gly Glu Thr 50 55 60Tyr Asn Gly Glu Ala Asn Asp Asp Glu Gly Ser Ser Glu Ala Thr Glu65 70 75 80Gly His Asp Glu Asp Asp Glu Ile Tyr Glu Gly Glu Tyr Gln Gly Ile 85 90 95Pro Ser Met Asn Gln Ala Lys Asp Ser Ile Val Ser Val Gly Gln Pro 100 105 110Lys Gly Asp Glu Tyr Lys Asp Arg Arg Glu Leu Glu Ser Glu Arg Arg 115 120 125Ala Asp Glu Glu Glu Leu Ala Gln Gln Tyr Glu Leu Ile Ile Gln Glu 130 135 140Cys Gly His Gly Arg Phe Gln Trp Ala Leu Phe Phe Val Leu Gly Met145 150 155 160Ala Leu Met Ala Asp Gly Val Glu Val Phe Val Val Gly Phe Val Leu 165 170 175Pro Ser Ala Glu Thr Asp Leu Cys Ile Pro Asn Ser Gly Ser Gly Trp 180 185 190Leu Gly Ser Ile Val Tyr Leu Gly Met Met Val Gly Ala Phe Phe Trp 195 200 205Gly Gly Leu Ala Asp Lys Val Gly Arg Lys Gln Ser Leu Leu Ile Cys 210 215 220Met Ser Val Asn Gly Phe Phe Ala Phe Leu Ser Ser Phe Val Gln Gly225 230 235 240Tyr Gly Phe Phe Leu Phe Cys Arg Leu Leu Ser Gly Phe Gly Ile Gly 245 250 255Gly Ala Ile Pro Thr Val Phe Ser Tyr Phe Ala Glu Val Leu Ala Arg 260 265 270Glu Lys Arg Gly Glu His Leu Ser Trp Leu Cys Met Phe Trp Met Ile 275 280 285Gly Gly Ile Tyr Ala Ser Ala Met Ala Trp Ala Ile Ile Pro His Tyr 290 295 300Gly Trp Ser Phe Ser Met Gly Ser Ala Tyr Gln Phe His Ser Trp Arg305 310 315 320Val Phe Val Ile Val Cys Ala Leu Pro Cys Val Ser Ser Val Val Ala 325 330 335Leu Thr Phe Met Pro Glu Ser Pro Arg Phe Leu Leu Glu Val Gly Lys 340 345 350His Asp Glu Ala Trp Met Ile Leu Lys Leu Ile His Asp Thr Asn Met 355 360 365Arg Ala Arg Gly Gln Pro Glu Lys Val Phe Thr Val Asn Lys Ile Lys 370 375 380Thr Pro Lys Gln Ile Asp Glu Leu Ile Glu Ile Glu Ser Asp Thr Gly385 390 395 400Thr Trp Tyr Arg Arg Cys Phe Val Arg Ile Arg Thr Glu Leu Tyr Gly 405 410 415Ile Trp Leu Thr Phe Met Arg Cys Phe Asn Tyr Pro Val Arg Asp Asn 420 425 430Thr Ile Lys Leu Thr Ile Val Trp Phe Thr Leu Ser Phe Gly Tyr Tyr 435 440 445Gly Leu Ser Val Trp Phe Pro Asp Val Ile Lys Pro Leu Gln Ser Asp 450 455 460Glu Tyr Ala Leu Leu Thr Arg Asn Val Glu Arg Asp Lys Tyr Ala Asn465 470 475 480Phe Thr Ile Asn Phe Thr Met Glu Asn Gln Ile His Thr Gly Met Glu 485 490 495Tyr Asp Asn Gly Arg Phe Ile Gly Val Lys Phe Lys Ser Val Thr Phe 500 505 510Lys Asp Ser Val Phe Lys Ser Cys Thr Phe Glu Asp Val Thr Ser Val 515 520 525Asn Thr Tyr Phe Lys Asn Cys Thr Phe Ile Asp Thr Val Phe Asp Asn 530 535 540Thr Asp Phe Glu Pro Tyr Lys Phe Ile Asp Ser Glu Phe Lys Asn Cys545 550 555 560Ser Phe Phe His Asn Lys Thr Gly Cys Gln Ile Thr Phe Asp Asp Asp 565 570 575Tyr Ser Ala Tyr Trp Ile Tyr Phe Val Asn Phe Leu Gly Thr Leu Ala 580 585 590Val Leu Pro Gly Asn Ile Val Ser Ala Leu Leu Met Asp Arg Ile Gly 595 600 605Arg Leu Thr Met Leu Gly Gly Ser Met Val Leu Ser Gly Ile Ser Cys 610 615 620Phe Phe Leu Trp Phe Gly Thr Ser Glu Ser Met Met Ile Gly Met Leu625 630 635 640Cys Leu Tyr Asn Gly Leu Thr Ile Ser Ala Trp Asn Ser Leu Asp Val 645 650 655Val Thr Val Glu Leu Tyr Pro Thr Asp Arg Arg Ala Thr Gly Phe Gly 660 665 670Phe Leu Asn Ala Leu Cys Lys Ala Ala Ala Val Leu Gly Asn Leu Ile 675 680 685Phe Gly Ser Leu Val Ser Ile Thr Lys Ser Ile Pro Ile Leu Leu Ala 690 695 700Ser Thr Val Leu Val Cys Gly Gly Leu Val Gly Leu Cys Leu Pro Asp705 710 715 720Thr Arg Thr Gln Val Leu Met 72531742PRTHomo sapiens 31Met Glu Glu Gly Phe Arg Asp Arg Ala Ala Phe Ile Arg Gly Ala Lys1 5 10 15Asp Ile Ala Lys Glu Val Lys Lys His Ala Ala Lys Lys Val Val Lys 20 25 30Gly Leu Asp Arg Val Gln Asp Glu Tyr Ser Arg Arg Ser Tyr Ser Arg 35 40 45Phe Glu Glu Glu Asp Asp Asp Asp Asp Phe Pro Ala Pro Ser Asp Gly 50 55 60Tyr Tyr Arg Gly Glu Gly Thr Gln Asp Glu Glu Glu Gly Gly Ala Ser65 70 75 80Ser Asp Ala Thr Glu Gly His Asp Glu Asp Asp Glu Ile Tyr Glu Gly 85 90 95Glu Tyr Gln Asp Ile Pro Arg Ala Glu Ser Gly Gly Lys Gly Glu Arg 100 105 110Met Ala Asp Gly Ala Pro Leu Ala Gly Val Arg Gly Gly Leu Ser Asp 115 120 125Gly Glu Gly Pro Pro Gly Gly Arg Gly Glu Ala Gln Arg Arg Lys Glu 130 135 140Arg Glu Glu Leu Ala Gln Gln Tyr Glu Ala Ile Leu Arg Glu Cys Gly145 150 155 160His Gly Arg Phe Gln Trp Thr Leu Tyr Phe Val Leu Gly Leu Ala Leu 165 170 175Met Ala Asp Gly Val Glu Val Phe Val Val Gly Phe Val Leu Pro Ser 180 185 190Ala Glu Lys Asp Met Cys Leu Ser Asp Ser Asn Lys Gly Met Leu Gly 195 200 205Leu Ile Val Tyr Leu Gly Met Met Val Gly Ala Phe Leu Trp Gly Gly 210 215 220Leu Ala Asp Arg Leu Gly Arg Arg Gln Cys Leu Leu Ile Ser Leu Ser225 230 235 240Val Asn Ser Val Phe Ala Phe Phe Ser Ser Phe Val Gln Gly Tyr Gly 245 250 255Thr Phe Leu Phe Cys Arg Leu Leu Ser Gly Val Gly Ile Gly Gly Ser 260 265 270Ile Pro Ile Val Phe Ser Tyr Phe Ser Glu Phe Leu Ala Gln Glu Lys 275 280 285Arg Gly Glu His Leu Ser Trp Leu Cys Met Phe Trp Met Ile Gly Gly 290 295 300Val Tyr Ala Ala Ala Met Ala Trp Ala Ile Ile Pro His Tyr Gly Trp305 310 315 320Ser Phe Gln Met Gly Ser Ala Tyr Gln Phe His Ser Trp Arg Val Phe 325 330 335Val Leu Val Cys Ala Phe Pro Ser Val Phe Ala Ile Gly Ala Leu Thr 340 345 350Thr Gln Pro Glu Ser Pro Arg Phe Phe Leu Glu Asn Gly Lys His Asp 355 360 365Glu Ala Trp Met Val Leu Lys Gln Val His Asp Thr Asn Met Arg Ala 370 375 380Lys Gly His Pro Glu Arg Val Phe Ser Val Thr His Ile Lys Thr Ile385 390 395 400His Gln Glu Asp Glu Leu Ile Glu Ile Gln Ser Asp Thr Gly Thr Trp 405 410 415Tyr Gln Arg Trp Gly Val Arg Ala Leu Ser Leu Gly Gly Gln Val Trp 420 425 430Gly Asn Phe Leu Ser Cys Phe Gly Pro Glu Tyr Arg Arg Ile Thr Leu 435 440 445Met Met Met Gly Val Trp Phe Thr Met Ser Phe Ser Tyr Tyr Gly Leu 450 455 460Thr Val Trp Phe Pro Asp Met Ile Arg His Leu Gln Ala Val Asp Tyr465 470 475 480Ala Ser Arg Thr Lys Val Phe Pro Gly Glu Arg Val Gly His Val Thr 485 490 495Phe Asn Phe Thr Leu Glu Asn Gln Ile His Arg Gly Gly Gln Tyr Phe 500 505 510Asn Asp Lys Phe Ile Gly Leu Arg Leu Lys Ser Val Ser Phe Glu Asp 515 520 525Ser Leu Phe Glu Glu Cys Tyr Phe Glu Asp Val Thr Ser Ser Asn Thr 530 535 540Phe Phe Arg Asn Cys Thr Phe Ile Asn Thr Val Phe Tyr Asn Thr Asp545 550 555 560Leu Phe Glu Tyr Lys Phe Val Asn Ser Arg Leu Ile Asn Ser Thr Phe 565 570 575Leu His Asn Lys Glu Gly Cys Pro Leu Asp Val Thr Gly Thr Gly Glu 580 585 590Gly Ala Tyr Met Val Tyr Phe Val Ser Phe Leu Gly Thr Leu Ala Val 595 600 605Leu Pro Gly Asn Ile Val Ser Ala Leu Leu Met Asp Lys Ile Gly Arg 610 615 620Leu Arg Met Leu Ala Gly Ser Ser Val Met Ser Cys Val Ser Cys Phe625 630 635 640Phe Leu Ser Phe Gly Asn Ser Glu Ser Ala Met Ile Ala Leu Leu Cys 645 650 655Leu Phe Gly Gly Val Ser Ile Ala Ser Trp Asn Ala Leu Asp Val Leu 660 665 670Thr Val Glu Leu Tyr Pro Ser Asp Lys Arg Thr Thr Ala Phe Gly Phe 675 680 685Leu Asn Ala Leu Cys Lys Leu Ala Ala Val Leu Gly Ile Ser Ile Phe 690 695 700Thr Ser Phe Val Gly Ile Thr Lys Ala Ala Pro Ile Leu Phe Ala Ser705 710 715 720Ala Ala Leu Ala Leu Gly Ser Ser Leu Ala Leu Lys Leu Pro Glu Thr 725 730 735Arg Gly Gln Val Leu Gln 740327PRTArtificial SequenceSNAP-25 antigen having a free carboxyl-terminus at the P1 residue of the scissile bond of the BoNT/A cleavage site 32Arg Ile Asp Glu Ala Asn Gln1 5338PRTArtificial SequenceSNAP-25 antigen having a free carboxyl-terminus at the P1 residue of the scissile bond of the BoNT/A cleavage site 33Thr Arg Ile Asp Glu Ala Asn Gln1 5349PRTArtificial SequenceSNAP-25 antigen having a free carboxyl-terminus at the P1 residue of the scissile bond of the BoNT/A cleavage site 34Lys Thr Arg Ile Asp Glu Ala Asn Gln1 53510PRTArtificial SequenceSNAP-25 antigen having a free carboxyl-terminus at the P1 residue of the scissile bond of the BoNT/A cleavage site 35Asn Lys Thr Arg Ile Asp Glu Ala Asn Gln1 5 103611PRTArtificial SequenceSNAP-25 antigen having a free carboxyl-terminus at the P1 residue of the scissile bond of the BoNT/A cleavage site 36Ser Asn Lys Thr Arg Ile Asp Glu Ala Asn Gln1 5 103712PRTArtificial SequenceSNAP-25 antigen having a free carboxyl-terminus at the P1 residue of the scissile bond of the BoNT/A cleavage site 37Asp Ser Asn Lys Thr Arg Ile Asp Glu Ala Asn Gln1 5 103813PRTArtificial SequenceSNAP-25 antigen having a free carboxylated carboxyl-terminus at the P1 residue of the scissile bond of the BoNT/A cleavage site 38Cys Asp Ser Asn Lys Thr Arg Ile Asp Glu Ala Asn Gln1 5 10397PRTArtificial SequenceSNAP-25 antigen having a free carboxyl-terminus at the P1 residue of the scissile bond of the BoNT/A cleavage site 39Arg Ile Asp Glu Ala Asn Lys1 5408PRTArtificial SequenceSNAP-25 antigen having a free carboxyl-terminus at the P1 residue of the scissile bond of the BoNT/A cleavage site 40Ala Arg Ile Asp Glu Ala Asn Lys1 5419PRTArtificial SequenceSNAP-25 antigen having a free carboxyl-terminus at the P1 residue of the scissile bond of the BoNT/A cleavage site 41Lys Ala Arg Ile Asp Glu Ala Asn Lys1 54210PRTArtificial SequenceSNAP-25 antigen having a free carboxyl-terminus at the P1 residue of the scissile bond of the BoNT/A cleavage site 42Asn Lys Ala Arg Ile Asp Glu Ala Asn Lys1 5 104311PRTArtificial SequenceSNAP-25 antigen

having a free carboxyl-terminus at the P1 residue of the scissile bond of the BoNT/A cleavage site 43Met Asn Lys Ala Arg Ile Asp Glu Ala Asn Lys1 5 104412PRTArtificial SequenceSNAP-25 antigen having a free carboxyl-terminus at the P1 residue of the scissile bond of the BoNT/A cleavage site 44Asp Met Asn Lys Ala Arg Ile Asp Glu Ala Asn Lys1 5 104513PRTArtificial SequenceSNAP-25 antigen having a free carboxylated carboxyl-terminus at the P1 residue of the scissile bond of the BoNT/A cleavage site 45Cys Asp Met Asn Lys Ala Arg Ile Asp Glu Ala Asn Lys1 5 104611PRTArtificial SequenceSNAP-25 antigen 46Cys Gly Gly Gly Arg Ile Asp Glu Ala Asn Gln1 5 104711PRTArtificial SequenceSNAP-25 antigen 47Cys Gly Gly Gly Arg Ile Asp Glu Ala Asn Lys1 5 104888PRTArtificial SequenceBirA-HisTag?-SNAP-25-134-197 48Met Gly Gly Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp1 5 10 15His His His His His His His His Ile Arg Arg Val Thr Asn Asp Ala 20 25 30Arg Glu Asn Glu Met Asp Glu Asn Leu Glu Gln Val Ser Gly Ile Ile 35 40 45Gly Asn Leu Arg His Met Ala Leu Asp Met Gly Asn Glu Ile Asp Thr 50 55 60Gln Asn Arg Gln Ile Asp Arg Ile Met Glu Lys Ala Asp Ser Asn Lys65 70 75 80Thr Arg Ile Asp Glu Ala Asn Gln 854997PRTArtificial SequenceBirA-HisTag?-SNAP-25-134-206 49Met Gly Gly Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp1 5 10 15His His His His His His His His Ile Arg Arg Val Thr Asn Asp Ala 20 25 30Arg Glu Asn Glu Met Asp Glu Asn Leu Glu Gln Val Ser Gly Ile Ile 35 40 45Gly Asn Leu Arg His Met Ala Leu Asp Met Gly Asn Glu Ile Asp Thr 50 55 60Gln Asn Arg Gln Ile Asp Arg Ile Met Glu Lys Ala Asp Ser Asn Lys65 70 75 80Thr Arg Ile Asp Glu Ala Asn Gln Arg Ala Thr Lys Met Leu Gly Ser 85 90 95Gly5016PRTArtificial SequenceBirA peptide 50Gly Gly Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp His1 5 10 15517570DNAArtificial SequencepQBI-25/GFP-BoNT/A-LC expression construct. 51gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg 60ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180ttagggttag gcgttttgcg ctgcttcgcc tcgaggcctg gccattgcat acgttgtatc 240catatcataa tatgtacatt tatattggct catgtccaac attaccgcca tgttgacatt 300gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 360tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 420cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 480attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 540atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 600atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 660tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 720actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 780aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 840gtaggcgtgt acggtgggag gtctatataa gcagagctcg tttagtgaac cgtcagatcg 900cctggagacg ccatccacgc tgttttgacc tccatagaag acaccgggac cgatccagcc 960tccgcgggcc accatggagg gcccggttac cggtaccgga tccagatatc tgggcggccg 1020ctcagcaagc ttcgcgaatt cgggaggcgg aggtggagct agcaaaggag aagaactctt 1080cactggagtt gtcccaattc ttgttgaatt agatggtgat gttaacggcc acaagttctc 1140tgtcagtgga gagggtgaag gtgatgcaac atacggaaaa cttaccctga agttcatctg 1200cactactggc aaactgcctg ttccatggcc aacactagtc actactctgt gctatggtgt 1260tcaatgcttt tcaagatacc cggatcatat gaaacggcat gactttttca agagtgccat 1320gcccgaaggt tatgtacagg aaaggaccat cttcttcaaa gatgacggca actacaagac 1380acgtgctgaa gtcaagtttg aaggtgatac ccttgttaat agaatcgagt taaaaggtat 1440tgacttcaag gaagatggca acattctggg acacaaattg gaatacaact ataactcaca 1500caatgtatac atcatggcag acaaacaaaa gaatggaatc aaagtgaact tcaagacccg 1560ccacaacatt gaagatggaa gcgttcaact agcagaccat tatcaacaaa atactccaat 1620tggcgatggc cctgtccttt taccagacaa ccattacctg tccacacaat ctgccctttc 1680gaaagatccc aacgaaaaga gagaccacat ggtccttctt gagtttgtaa cagctgctgg 1740gattacacat ggcatggatg aactgtacaa catcgatgga ggcggaggtg gaccttttgt 1800taataaacaa tttaattata aagatcctgt aaatggtgtt gatattgctt atataaaaat 1860tccaaatgca ggacaaatgc aaccagtaaa agcttttaaa attcataata aaatatgggt 1920tattccagaa agagatacat ttacaaatcc tgaagaagga gatttaaatc caccaccaga 1980agcaaaacaa gttccagttt catattatga ttcaacatat ttaagtacag ataatgaaaa 2040agataattat ttaaagggag ttacaaaatt atttgagaga atttattcaa ctgatcttgg 2100aagaatgttg ttaacatcaa tagtaagggg aataccattt tggggtggaa gtacaataga 2160tacagaatta aaagttattg atactaattg tattaatgtg atacaaccag atggtagtta 2220tagatcagaa gaacttaatc tagtaataat aggaccctca gctgatatta tacagtttga 2280atgtaaaagc tttggacatg aagttttgaa tcttacgcga aatggttatg gctctactca 2340atacattaga tttagcccag attttacatt tggttttgag gagtcacttg aagttgatac 2400aaatcctctt ttaggtgcag gcaaatttgc tacagatcca gcagtaacat tagcacatga 2460acttatacat gctggacata gattatatgg aatagcaatt aatccaaata gggtttttaa 2520agtaaatact aatgcctatt atgaaatgag tgggttagaa gtaagctttg aggaacttag 2580aacatttggg ggacatgatg caaagtttat agatagttta caggaaaacg aatttcgtct 2640atattattat aataagttta aagatatagc aagtacactt aataaagcta aatcaatagt 2700aggtactact gcttcattac agtatatgaa aaatgttttt aaagagaaat atctcctatc 2760tgaagataca tctggaaaat tttcggtaga taaattaaaa tttgataagt tatacaaaat 2820gttaacagag atttacacag aggataattt tgttaagttt tttaaagtac ttaacagaaa 2880aacatatttg aattttgata aagccgtatt taagataaat atagtaccta aggtaaatta 2940cacaatatat gatggattta atttaagaaa tacaaattta gcagcaaact ttaatggtca 3000aaatacagaa attaataata tgaattttac taaactaaaa aattttactg gattgtttga 3060attttataag ttgctatgtg taagagggat aatcacttcg aaatgaacgc gttggcccta 3120ttctatagtg tcacctaaat gctagagctc gctgatcagc ctcgactgtg ccttctagtt 3180gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc 3240ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt 3300ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca 3360ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct 3420ctagggggta tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta 3480cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc 3540cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg ggcatccctt 3600tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg 3660gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca 3720cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct 3780attcttttga tttataaggg attttgggga tttcggccta ttggttaaaa aatgagctga 3840tttaacaaaa atttaacgcg aattaattct gtggaatgtg tgtcagttag ggtgtggaaa 3900gtccccaggc tccccaggca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa 3960ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca 4020attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca 4080gttccgccca ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg 4140ccgcctctgc ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct 4200tttgcaaaaa gctcccggga gcttgtatat ccattttcgg atctgatcaa gagacaggat 4260gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg 4320tggagaggct attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg 4380tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg 4440ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc 4500cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg 4560aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca 4620tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc 4680aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg 4740atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg 4800cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata 4860tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg 4920accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat 4980gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct 5040tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa tgaccgacca 5100agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct atgaaaggtt 5160gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat 5220gctggagttc ttcgcccacc ccaacttgtt tattgcagct tataatggtt acaaataaag 5280caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt 5340gtccaaactc atcaatgtat cttatcatgt ctgtataccg tcgacctcta gctagagctt 5400ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca 5460caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact 5520cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct 5580gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc 5640ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca 5700ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg 5760agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca 5820taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa 5880cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc 5940tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc 6000gctttctcaa tgctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct 6060gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg 6120tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag 6180gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta 6240cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg 6300aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt 6360tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt 6420ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag 6480attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat 6540ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc 6600tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat 6660aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc 6720acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag 6780aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag 6840agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt 6900ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg 6960agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt 7020tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc 7080tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc 7140attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa 7200taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg 7260aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc 7320caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag 7380gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt 7440cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt 7500tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc 7560acctgacgtc 757052682PRTArtificial SequenceGFP-BoNT/A light chain amino acid sequence. 52Ala Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val1 5 10 15Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu 20 25 30Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys 35 40 45Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu 50 55 60Cys Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Arg65 70 75 80His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg 85 90 95Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val 100 105 110Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile 115 120 125Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn 130 135 140Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly145 150 155 160Ile Lys Val Asn Phe Lys Thr Arg His Asn Ile Glu Asp Gly Ser Val 165 170 175Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro 180 185 190Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser 195 200 205Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val 210 215 220Thr Ala Ala Gly Ile Thr His Gly Met Asp Glu Leu Tyr Asn Ile Asp225 230 235 240Gly Gly Gly Gly Gly Pro Phe Val Asn Lys Gln Phe Asn Tyr Lys Asp 245 250 255Pro Val Asn Gly Val Asp Ile Ala Tyr Ile Lys Ile Pro Asn Ala Gly 260 265 270Gln Met Gln Pro Val Lys Ala Phe Lys Ile His Asn Lys Ile Trp Val 275 280 285Ile Pro Glu Arg Asp Thr Phe Thr Asn Pro Glu Glu Gly Asp Leu Asn 290 295 300Pro Pro Pro Glu Ala Lys Gln Val Pro Val Ser Tyr Tyr Asp Ser Thr305 310 315 320Tyr Leu Ser Thr Asp Asn Glu Lys Asp Asn Tyr Leu Lys Gly Val Thr 325 330 335Lys Leu Phe Glu Arg Ile Tyr Ser Thr Asp Leu Gly Arg Met Leu Leu 340 345 350Thr Ser Ile Val Arg Gly Ile Pro Phe Trp Gly Gly Ser Thr Ile Asp 355 360 365Thr Glu Leu Lys Val Ile Asp Thr Asn Cys Ile Asn Val Ile Gln Pro 370 375 380Asp Gly Ser Tyr Arg Ser Glu Glu Leu Asn Leu Val Ile Ile Gly Pro385 390 395 400Ser Ala Asp Ile Ile Gln Phe Glu Cys Lys Ser Phe Gly His Glu Val 405 410 415Leu Asn Leu Thr Arg Asn Gly Tyr Gly Ser Thr Gln Tyr Ile Arg Phe 420 425 430Ser Pro Asp Phe Thr Phe Gly Phe Glu Glu Ser Leu Glu Val Asp Thr 435 440 445Asn Pro Leu Leu Gly Ala Gly Lys Phe Ala Thr Asp Pro Ala Val Thr 450 455 460Leu Ala His Glu Leu Ile His Ala Gly His Arg Leu Tyr Gly Ile Ala465 470 475 480Ile Asn Pro Asn Arg Val Phe Lys Val Asn Thr Asn Ala Tyr Tyr Glu 485 490 495Met Ser Gly Leu Glu Val Ser Phe Glu Glu Leu Arg Thr Phe Gly Gly 500 505 510His Asp Ala Lys Phe Ile Asp Ser Leu Gln Glu Asn Glu Phe Arg Leu 515 520 525Tyr Tyr Tyr Asn Lys Phe Lys Asp Ile Ala Ser Thr Leu Asn Lys Ala 530 535 540Lys Ser Ile Val Gly Thr Thr Ala Ser Leu Gln Tyr Met Lys Asn Val545 550 555 560Phe Lys Glu Lys Tyr Leu Leu Ser Glu Asp Thr Ser Gly Lys Phe Ser 565 570 575Val Asp Lys Leu Lys Phe Asp Lys Leu Tyr Lys Met Leu Thr Glu Ile 580 585 590Tyr Thr Glu Asp Asn Phe Val Lys Phe Phe Lys Val Leu Asn Arg Lys 595 600 605Thr Tyr Leu Asn Phe Asp Lys Ala Val Phe Lys Ile Asn Ile Val Pro 610 615 620Lys Val Asn Tyr Thr Ile Tyr Asp Gly Phe Asn Leu Arg Asn Thr Asn625 630 635 640Leu Ala Ala Asn Phe Asn Gly Gln Asn Thr Glu Ile Asn Asn Met Asn 645 650 655Phe Thr Lys Leu Lys Asn Phe Thr Gly Leu Phe Glu Phe Tyr Lys Leu 660 665 670Leu Cys Val Arg Gly Ile Ile Thr Ser Lys 675 680536259DNAArtificial SequencepQBI-25/GFP expression construct. 53gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg 60ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180ttagggttag gcgttttgcg ctgcttcgcc tcgaggcctg gccattgcat acgttgtatc 240catatcataa tatgtacatt tatattggct catgtccaac attaccgcca tgttgacatt 300gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 360tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 420cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 480attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 540atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 600atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 660tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 720actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 780aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 840gtaggcgtgt acggtgggag gtctatataa gcagagctcg tttagtgaac cgtcagatcg 900cctggagacg ccatccacgc tgttttgacc tccatagaag

acaccgggac cgatccagcc 960tccgcgggcc accatggagg gcccggttac cggtaccgga tccagatatc tgggcggccg 1020ctcagcaagc ttcgcgaatt cgggaggcgg aggtggagct agcaaaggag aagaactctt 1080cactggagtt gtcccaattc ttgttgaatt agatggtgat gttaacggcc acaagttctc 1140tgtcagtgga gagggtgaag gtgatgcaac atacggaaaa cttaccctga agttcatctg 1200cactactggc aaactgcctg ttccatggcc aacactagtc actactctgt gctatggtgt 1260tcaatgcttt tcaagatacc cggatcatat gaaacggcat gactttttca agagtgccat 1320gcccgaaggt tatgtacagg aaaggaccat cttcttcaaa gatgacggca actacaagac 1380acgtgctgaa gtcaagtttg aaggtgatac ccttgttaat agaatcgagt taaaaggtat 1440tgacttcaag gaagatggca acattctggg acacaaattg gaatacaact ataactcaca 1500caatgtatac atcatggcag acaaacaaaa gaatggaatc aaagtgaact tcaagacccg 1560ccacaacatt gaagatggaa gcgttcaact agcagaccat tatcaacaaa atactccaat 1620tggcgatggc cctgtccttt taccagacaa ccattacctg tccacacaat ctgccctttc 1680gaaagatccc aacgaaaaga gagaccacat ggtccttctt gagtttgtaa cagctgctgg 1740gattacacat ggcatggatg aactgtacaa catcgatgga ggcggaggtg gatgaacgcg 1800ttggccctat tctatagtgt cacctaaatg ctagagctcg ctgatcagcc tcgactgtgc 1860cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag 1920gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta 1980ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag 2040acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca 2100gctggggctc tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg 2160tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg 2220ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg 2280gcatcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt 2340agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt 2400tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta 2460tctcggtcta ttcttttgat ttataaggga ttttggggat ttcggcctat tggttaaaaa 2520atgagctgat ttaacaaaaa tttaacgcga attaattctg tggaatgtgt gtcagttagg 2580gtgtggaaag tccccaggct ccccaggcag gcagaagtat gcaaagcatg catctcaatt 2640agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca 2700tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa 2760ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag 2820aggccgaggc cgcctctgcc tctgagctat tccagaagta gtgaggaggc ttttttggag 2880gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc cattttcgga tctgatcaag 2940agacaggatg aggatcgttt cgcatgattg aacaagatgg attgcacgca ggttctccgg 3000ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg 3060atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc 3120tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga 3180cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc 3240tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag 3300tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat 3360tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg 3420tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca 3480ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt gacccatggc gatgcctgct 3540tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg 3600gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg 3660gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc 3720gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc gggactctgg ggttcgaaat 3780gaccgaccaa gcgacgccca acctgccatc acgagatttc gattccaccg ccgccttcta 3840tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg 3900ggatctcatg ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta 3960caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag 4020ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag 4080ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac 4140aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt 4200gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc 4260gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg 4320ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt 4380atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa 4440gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc 4500gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag 4560gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt 4620gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg 4680aagcgtggcg ctttctcaat gctcacgctg taggtatctc agttcggtgt aggtcgttcg 4740ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg 4800taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac 4860tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg 4920gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt 4980taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg 5040tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc 5100tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt 5160ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt 5220taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag 5280tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt 5340cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc 5400gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc 5460cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg 5520ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac 5580aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg 5640atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc 5700tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact 5760gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc 5820aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat 5880acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc 5940ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac 6000tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa 6060aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact 6120catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg 6180atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg 6240aaaagtgcca cctgacgtc 625954245PRTArtificial SequenceGFP amino acid sequence. 54Ala Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val1 5 10 15Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu 20 25 30Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys 35 40 45Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu 50 55 60Cys Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Arg65 70 75 80His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg 85 90 95Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val 100 105 110Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile 115 120 125Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn 130 135 140Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly145 150 155 160Ile Lys Val Asn Phe Lys Thr Arg His Asn Ile Glu Asp Gly Ser Val 165 170 175Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro 180 185 190Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser 195 200 205Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val 210 215 220Thr Ala Ala Gly Ile Thr His Gly Met Asp Glu Leu Tyr Asn Ile Asp225 230 235 240Gly Gly Gly Gly Gly 245554PRTArtificial SequenceG-spacer flexible spacer 55Gly Gly Gly Gly1565PRTArtificial SequenceG-spacer flexible spacer 56Gly Gly Gly Gly Ser1 5574PRTArtificial SequenceA-spacer flexible spacer 57Ala Ala Ala Ala1585PRTArtificial SequenceA-spacer flexible spacer 58Ala Ala Ala Ala Val1 559821PRTHomo sapiens 59Met Val Ser Trp Gly Arg Phe Ile Cys Leu Val Val Val Thr Met Ala1 5 10 15Thr Leu Ser Leu Ala Arg Pro Ser Phe Ser Leu Val Glu Asp Thr Thr 20 25 30Leu Glu Pro Glu Glu Pro Pro Thr Lys Tyr Gln Ile Ser Gln Pro Glu 35 40 45Val Tyr Val Ala Ala Pro Gly Glu Ser Leu Glu Val Arg Cys Leu Leu 50 55 60Lys Asp Ala Ala Val Ile Ser Trp Thr Lys Asp Gly Val His Leu Gly65 70 75 80Pro Asn Asn Arg Thr Val Leu Ile Gly Glu Tyr Leu Gln Ile Lys Gly 85 90 95Ala Thr Pro Arg Asp Ser Gly Leu Tyr Ala Cys Thr Ala Ser Arg Thr 100 105 110Val Asp Ser Glu Thr Trp Tyr Phe Met Val Asn Val Thr Asp Ala Ile 115 120 125Ser Ser Gly Asp Asp Glu Asp Asp Thr Asp Gly Ala Glu Asp Phe Val 130 135 140Ser Glu Asn Ser Asn Asn Lys Arg Ala Pro Tyr Trp Thr Asn Thr Glu145 150 155 160Lys Met Glu Lys Arg Leu His Ala Val Pro Ala Ala Asn Thr Val Lys 165 170 175Phe Arg Cys Pro Ala Gly Gly Asn Pro Met Pro Thr Met Arg Trp Leu 180 185 190Lys Asn Gly Lys Glu Phe Lys Gln Glu His Arg Ile Gly Gly Tyr Lys 195 200 205Val Arg Asn Gln His Trp Ser Leu Ile Met Glu Ser Val Val Pro Ser 210 215 220Asp Lys Gly Asn Tyr Thr Cys Val Val Glu Asn Glu Tyr Gly Ser Ile225 230 235 240Asn His Thr Tyr His Leu Asp Val Val Glu Arg Ser Pro His Arg Pro 245 250 255Ile Leu Gln Ala Gly Leu Pro Ala Asn Ala Ser Thr Val Val Gly Gly 260 265 270Asp Val Glu Phe Val Cys Lys Val Tyr Ser Asp Ala Gln Pro His Ile 275 280 285Gln Trp Ile Lys His Val Glu Lys Asn Gly Ser Lys Tyr Gly Pro Asp 290 295 300Gly Leu Pro Tyr Leu Lys Val Leu Lys Ala Ala Gly Val Asn Thr Thr305 310 315 320Asp Lys Glu Ile Glu Val Leu Tyr Ile Arg Asn Val Thr Phe Glu Asp 325 330 335Ala Gly Glu Tyr Thr Cys Leu Ala Gly Asn Ser Ile Gly Ile Ser Phe 340 345 350His Ser Ala Trp Leu Thr Val Leu Pro Ala Pro Gly Arg Glu Lys Glu 355 360 365Ile Thr Ala Ser Pro Asp Tyr Leu Glu Ile Ala Ile Tyr Cys Ile Gly 370 375 380Val Phe Leu Ile Ala Cys Met Val Val Thr Val Ile Leu Cys Arg Met385 390 395 400Lys Asn Thr Thr Lys Lys Pro Asp Phe Ser Ser Gln Pro Ala Val His 405 410 415Lys Leu Thr Lys Arg Ile Pro Leu Arg Arg Gln Val Thr Val Ser Ala 420 425 430Glu Ser Ser Ser Ser Met Asn Ser Asn Thr Pro Leu Val Arg Ile Thr 435 440 445Thr Arg Leu Ser Ser Thr Ala Asp Thr Pro Met Leu Ala Gly Val Ser 450 455 460Glu Tyr Glu Leu Pro Glu Asp Pro Lys Trp Glu Phe Pro Arg Asp Lys465 470 475 480Leu Thr Leu Gly Lys Pro Leu Gly Glu Gly Cys Phe Gly Gln Val Val 485 490 495Met Ala Glu Ala Val Gly Ile Asp Lys Asp Lys Pro Lys Glu Ala Val 500 505 510Thr Val Ala Val Lys Met Leu Lys Asp Asp Ala Thr Glu Lys Asp Leu 515 520 525Ser Asp Leu Val Ser Glu Met Glu Met Met Lys Met Ile Gly Lys His 530 535 540Lys Asn Ile Ile Asn Leu Leu Gly Ala Cys Thr Gln Asp Gly Pro Leu545 550 555 560Tyr Val Ile Val Glu Tyr Ala Ser Lys Gly Asn Leu Arg Glu Tyr Leu 565 570 575Arg Ala Arg Arg Pro Pro Gly Met Glu Tyr Ser Tyr Asp Ile Asn Arg 580 585 590Val Pro Glu Glu Gln Met Thr Phe Lys Asp Leu Val Ser Cys Thr Tyr 595 600 605Gln Leu Ala Arg Gly Met Glu Tyr Leu Ala Ser Gln Lys Cys Ile His 610 615 620Arg Asp Leu Ala Ala Arg Asn Val Leu Val Thr Glu Asn Asn Val Met625 630 635 640Lys Ile Ala Asp Phe Gly Leu Ala Arg Asp Ile Asn Asn Ile Asp Tyr 645 650 655Tyr Lys Lys Thr Thr Asn Gly Arg Leu Pro Val Lys Trp Met Ala Pro 660 665 670Glu Ala Leu Phe Asp Arg Val Tyr Thr His Gln Ser Asp Val Trp Ser 675 680 685Phe Gly Val Leu Met Trp Glu Ile Phe Thr Leu Gly Gly Ser Pro Tyr 690 695 700Pro Gly Ile Pro Val Glu Glu Leu Phe Lys Leu Leu Lys Glu Gly His705 710 715 720Arg Met Asp Lys Pro Ala Asn Cys Thr Asn Glu Leu Tyr Met Met Met 725 730 735Arg Asp Cys Trp His Ala Val Pro Ser Gln Arg Pro Thr Phe Lys Gln 740 745 750Leu Val Glu Asp Leu Asp Arg Ile Leu Thr Leu Thr Thr Asn Glu Glu 755 760 765Tyr Leu Asp Leu Ser Gln Pro Leu Glu Gln Tyr Ser Pro Ser Tyr Pro 770 775 780Asp Thr Arg Ser Ser Cys Ser Ser Gly Asp Asp Ser Val Phe Ser Pro785 790 795 800Asp Pro Met Pro Tyr Glu Pro Cys Leu Pro Gln Tyr Pro His Ile Asn 805 810 815Gly Ser Val Lys Thr 82060822PRTHomo sapiens 60Met Val Ser Trp Gly Arg Phe Ile Cys Leu Val Val Val Thr Met Ala1 5 10 15Thr Leu Ser Leu Ala Arg Pro Ser Phe Ser Leu Val Glu Asp Thr Thr 20 25 30Leu Glu Pro Glu Glu Pro Pro Thr Lys Tyr Gln Ile Ser Gln Pro Glu 35 40 45Val Tyr Val Ala Ala Pro Gly Glu Ser Leu Glu Val Arg Cys Leu Leu 50 55 60Lys Asp Ala Ala Val Ile Ser Trp Thr Lys Asp Gly Val His Leu Gly65 70 75 80Pro Asn Asn Arg Thr Val Leu Ile Gly Glu Tyr Leu Gln Ile Lys Gly 85 90 95Ala Thr Pro Arg Asp Ser Gly Leu Tyr Ala Cys Thr Ala Ser Arg Thr 100 105 110Val Asp Ser Glu Thr Trp Tyr Phe Met Val Asn Val Thr Asp Ala Ile 115 120 125Ser Ser Gly Asp Asp Glu Asp Asp Thr Asp Gly Ala Glu Asp Phe Val 130 135 140Ser Glu Asn Ser Asn Asn Lys Arg Ala Pro Tyr Trp Thr Asn Thr Glu145 150 155 160Lys Met Glu Lys Arg Leu His Ala Val Pro Ala Ala Asn Thr Val Lys 165 170 175Phe Arg Cys Pro Ala Gly Gly Asn Pro Met Pro Thr Met Arg Trp Leu 180 185 190Lys Asn Gly Lys Glu Phe Lys Gln Glu His Arg Ile Gly Gly Tyr Lys 195 200 205Val Arg Asn Gln His Trp Ser Leu Ile Met Glu Ser Val Val Pro Ser 210 215 220Asp Lys Gly Asn Tyr Thr Cys Val Val Glu Asn Glu Tyr Gly Ser Ile225 230 235 240Asn His Thr Tyr His Leu Asp Val Val Glu Arg Ser Pro His Arg Pro 245 250 255Ile Leu Gln Ala Gly Leu Pro Ala Asn Ala Ser Thr Val Val Gly Gly 260 265 270Asp Val Glu Phe Val Cys Lys Val Tyr Ser Asp Ala Gln Pro His Ile 275 280 285Gln Trp Ile Lys His Val Glu Lys Asn Gly Ser Lys Tyr Gly Pro Asp 290 295 300Gly Leu Pro Tyr Leu Lys Val Leu Lys His Ser Gly Ile Asn Ser Ser305 310 315 320Asn Ala Glu Val Leu Ala Leu Phe Asn Val Thr Glu Ala Asp Ala Gly 325 330 335Glu Tyr Ile Cys Lys Val Ser Asn Tyr Ile Gly Gln Ala Asn Gln Ser 340 345 350Ala Trp Leu Thr Val Leu Pro Lys Gln Gln Ala Pro Gly Arg Glu Lys 355 360 365Glu Ile Thr Ala Ser Pro Asp Tyr Leu Glu Ile Ala Ile Tyr Cys Ile 370 375 380Gly Val Phe Leu Ile Ala Cys Met Val Val Thr Val Ile Leu Cys Arg385 390 395 400Met Lys Asn Thr Thr Lys Lys Pro Asp Phe Ser Ser Gln Pro Ala Val 405 410 415His Lys Leu Thr Lys Arg Ile Pro Leu Arg Arg Gln Val Thr Val Ser 420 425 430Ala Glu Ser Ser Ser Ser Met Asn Ser Asn Thr Pro Leu Val Arg Ile 435 440 445Thr Thr Arg Leu Ser Ser Thr Ala Asp Thr Pro Met Leu Ala Gly Val 450 455 460Ser Glu Tyr Glu Leu Pro Glu Asp Pro Lys Trp Glu

Phe Pro Arg Asp465 470 475 480Lys Leu Thr Leu Gly Lys Pro Leu Gly Glu Gly Cys Phe Gly Gln Val 485 490 495Val Met Ala Glu Ala Val Gly Ile Asp Lys Asp Lys Pro Lys Glu Ala 500 505 510Val Thr Val Ala Val Lys Met Leu Lys Asp Asp Ala Thr Glu Lys Asp 515 520 525Leu Ser Asp Leu Val Ser Glu Met Glu Met Met Lys Met Ile Gly Lys 530 535 540His Lys Asn Ile Ile Asn Leu Leu Gly Ala Cys Thr Gln Asp Gly Pro545 550 555 560Leu Tyr Val Ile Val Glu Tyr Ala Ser Lys Gly Asn Leu Arg Glu Tyr 565 570 575Leu Arg Ala Arg Arg Pro Pro Gly Met Glu Tyr Ser Tyr Asp Ile Asn 580 585 590Arg Val Pro Glu Glu Gln Met Thr Phe Lys Asp Leu Val Ser Cys Thr 595 600 605Tyr Gln Leu Ala Arg Gly Met Glu Tyr Leu Ala Ser Gln Lys Cys Ile 610 615 620His Arg Asp Leu Ala Ala Arg Asn Val Leu Val Thr Glu Asn Asn Val625 630 635 640Met Lys Ile Ala Asp Phe Gly Leu Ala Arg Asp Ile Asn Asn Ile Asp 645 650 655Tyr Tyr Lys Lys Thr Thr Asn Gly Arg Leu Pro Val Lys Trp Met Ala 660 665 670Pro Glu Ala Leu Phe Asp Arg Val Tyr Thr His Gln Ser Asp Val Trp 675 680 685Ser Phe Gly Val Leu Met Trp Glu Ile Phe Thr Leu Gly Gly Ser Pro 690 695 700Tyr Pro Gly Ile Pro Val Glu Glu Leu Phe Lys Leu Leu Lys Glu Gly705 710 715 720His Arg Met Asp Lys Pro Ala Asn Cys Thr Asn Glu Leu Tyr Met Met 725 730 735Met Arg Asp Cys Trp His Ala Val Pro Ser Gln Arg Pro Thr Phe Lys 740 745 750Gln Leu Val Glu Asp Leu Asp Arg Ile Leu Thr Leu Thr Thr Asn Glu 755 760 765Glu Tyr Leu Asp Leu Ser Gln Pro Leu Glu Gln Tyr Ser Pro Ser Tyr 770 775 780Pro Asp Thr Arg Ser Ser Cys Ser Ser Gly Asp Asp Ser Val Phe Ser785 790 795 800Pro Asp Pro Met Pro Tyr Glu Pro Cys Leu Pro Gln Tyr Pro His Ile 805 810 815Asn Gly Ser Val Lys Thr 82061769PRTHomo sapiens 61Met Val Ser Trp Gly Arg Phe Ile Cys Leu Val Val Val Thr Met Ala1 5 10 15Thr Leu Ser Leu Ala Arg Pro Ser Phe Ser Leu Val Glu Asp Thr Thr 20 25 30Leu Glu Pro Glu Glu Pro Pro Thr Lys Tyr Gln Ile Ser Gln Pro Glu 35 40 45Val Tyr Val Ala Ala Pro Gly Glu Ser Leu Glu Val Arg Cys Leu Leu 50 55 60Lys Asp Ala Ala Val Ile Ser Trp Thr Lys Asp Gly Val His Leu Gly65 70 75 80Pro Asn Asn Arg Thr Val Leu Ile Gly Glu Tyr Leu Gln Ile Lys Gly 85 90 95Ala Thr Pro Arg Asp Ser Gly Leu Tyr Ala Cys Thr Ala Ser Arg Thr 100 105 110Val Asp Ser Glu Thr Trp Tyr Phe Met Val Asn Val Thr Asp Ala Ile 115 120 125Ser Ser Gly Asp Asp Glu Asp Asp Thr Asp Gly Ala Glu Asp Phe Val 130 135 140Ser Glu Asn Ser Asn Asn Lys Arg Ala Pro Tyr Trp Thr Asn Thr Glu145 150 155 160Lys Met Glu Lys Arg Leu His Ala Val Pro Ala Ala Asn Thr Val Lys 165 170 175Phe Arg Cys Pro Ala Gly Gly Asn Pro Met Pro Thr Met Arg Trp Leu 180 185 190Lys Asn Gly Lys Glu Phe Lys Gln Glu His Arg Ile Gly Gly Tyr Lys 195 200 205Val Arg Asn Gln His Trp Ser Leu Ile Met Glu Ser Val Val Pro Ser 210 215 220Asp Lys Gly Asn Tyr Thr Cys Val Val Glu Asn Glu Tyr Gly Ser Ile225 230 235 240Asn His Thr Tyr His Leu Asp Val Val Glu Arg Ser Pro His Arg Pro 245 250 255Ile Leu Gln Ala Gly Leu Pro Ala Asn Ala Ser Thr Val Val Gly Gly 260 265 270Asp Val Glu Phe Val Cys Lys Val Tyr Ser Asp Ala Gln Pro His Ile 275 280 285Gln Trp Ile Lys His Val Glu Lys Asn Gly Ser Lys Tyr Gly Pro Asp 290 295 300Gly Leu Pro Tyr Leu Lys Val Leu Lys His Ser Gly Ile Asn Ser Ser305 310 315 320Asn Ala Glu Val Leu Ala Leu Phe Asn Val Thr Glu Ala Asp Ala Gly 325 330 335Glu Tyr Ile Cys Lys Val Ser Asn Tyr Ile Gly Gln Ala Asn Gln Ser 340 345 350Ala Trp Leu Thr Val Leu Pro Lys Gln Gln Ala Pro Gly Arg Glu Lys 355 360 365Glu Ile Thr Ala Ser Pro Asp Tyr Leu Glu Ile Ala Ile Tyr Cys Ile 370 375 380Gly Val Phe Leu Ile Ala Cys Met Val Val Thr Val Ile Leu Cys Arg385 390 395 400Met Lys Asn Thr Thr Lys Lys Pro Asp Phe Ser Ser Gln Pro Ala Val 405 410 415His Lys Leu Thr Lys Arg Ile Pro Leu Arg Arg Gln Val Thr Val Ser 420 425 430Ala Glu Ser Ser Ser Ser Met Asn Ser Asn Thr Pro Leu Val Arg Ile 435 440 445Thr Thr Arg Leu Ser Ser Thr Ala Asp Thr Pro Met Leu Ala Gly Val 450 455 460Ser Glu Tyr Glu Leu Pro Glu Asp Pro Lys Trp Glu Phe Pro Arg Asp465 470 475 480Lys Leu Thr Leu Gly Lys Pro Leu Gly Glu Gly Cys Phe Gly Gln Val 485 490 495Val Met Ala Glu Ala Val Gly Ile Asp Lys Asp Lys Pro Lys Glu Ala 500 505 510Val Thr Val Ala Val Lys Met Leu Lys Asp Asp Ala Thr Glu Lys Asp 515 520 525Leu Ser Asp Leu Val Ser Glu Met Glu Met Met Lys Met Ile Gly Lys 530 535 540His Lys Asn Ile Ile Asn Leu Leu Gly Ala Cys Thr Gln Asp Gly Pro545 550 555 560Leu Tyr Val Ile Val Glu Tyr Ala Ser Lys Gly Asn Leu Arg Glu Tyr 565 570 575Leu Arg Ala Arg Arg Pro Pro Gly Met Glu Tyr Ser Tyr Asp Ile Asn 580 585 590Arg Val Pro Glu Glu Gln Met Thr Phe Lys Asp Leu Val Ser Cys Thr 595 600 605Tyr Gln Leu Ala Arg Gly Met Glu Tyr Leu Ala Ser Gln Lys Cys Ile 610 615 620His Arg Asp Leu Ala Ala Arg Asn Val Leu Val Thr Glu Asn Asn Val625 630 635 640Met Lys Ile Ala Asp Phe Gly Leu Ala Arg Asp Ile Asn Asn Ile Asp 645 650 655Tyr Tyr Lys Lys Thr Thr Asn Gly Arg Leu Pro Val Lys Trp Met Ala 660 665 670Pro Glu Ala Leu Phe Asp Arg Val Tyr Thr His Gln Ser Asp Val Trp 675 680 685Ser Phe Gly Val Leu Met Trp Glu Ile Phe Thr Leu Gly Gly Ser Pro 690 695 700Tyr Pro Gly Ile Pro Val Glu Glu Leu Phe Lys Leu Leu Lys Glu Gly705 710 715 720His Arg Met Asp Lys Pro Ala Asn Cys Thr Asn Glu Leu Tyr Met Met 725 730 735Met Arg Asp Cys Trp His Ala Val Pro Ser Gln Arg Pro Thr Phe Lys 740 745 750Gln Leu Val Glu Asp Leu Asp Arg Ile Leu Thr Leu Thr Thr Asn Glu 755 760 765Ile62709PRTHomo sapiens 62Met Val Ser Trp Gly Arg Phe Ile Cys Leu Val Val Val Thr Met Ala1 5 10 15Thr Leu Ser Leu Ala Arg Pro Ser Phe Ser Leu Val Glu Asp Thr Thr 20 25 30Leu Glu Pro Glu Glu Pro Pro Thr Lys Tyr Gln Ile Ser Gln Pro Glu 35 40 45Val Tyr Val Ala Ala Pro Gly Glu Ser Leu Glu Val Arg Cys Leu Leu 50 55 60Lys Asp Ala Ala Val Ile Ser Trp Thr Lys Asp Gly Val His Leu Gly65 70 75 80Pro Asn Asn Arg Thr Val Leu Ile Gly Glu Tyr Leu Gln Ile Lys Gly 85 90 95Ala Thr Pro Arg Asp Ser Gly Leu Tyr Ala Cys Thr Ala Ser Arg Thr 100 105 110Val Asp Ser Glu Thr Trp Tyr Phe Met Val Asn Val Thr Asp Ala Ile 115 120 125Ser Ser Gly Asp Asp Glu Asp Asp Thr Asp Gly Ala Glu Asp Phe Val 130 135 140Ser Glu Asn Ser Asn Asn Lys Arg Ala Pro Tyr Trp Thr Asn Thr Glu145 150 155 160Lys Met Glu Lys Arg Leu His Ala Val Pro Ala Ala Asn Thr Val Lys 165 170 175Phe Arg Cys Pro Ala Gly Gly Asn Pro Met Pro Thr Met Arg Trp Leu 180 185 190Lys Asn Gly Lys Glu Phe Lys Gln Glu His Arg Ile Gly Gly Tyr Lys 195 200 205Val Arg Asn Gln His Trp Ser Leu Ile Met Glu Ser Val Val Pro Ser 210 215 220Asp Lys Gly Asn Tyr Thr Cys Val Val Glu Asn Glu Tyr Gly Ser Ile225 230 235 240Asn His Thr Tyr His Leu Asp Val Val Ala Pro Gly Arg Glu Lys Glu 245 250 255Ile Thr Ala Ser Pro Asp Tyr Leu Glu Ile Ala Ile Tyr Cys Ile Gly 260 265 270Val Phe Leu Ile Ala Cys Met Val Val Thr Val Ile Leu Cys Arg Met 275 280 285Lys Asn Thr Thr Lys Lys Pro Asp Phe Ser Ser Gln Pro Ala Val His 290 295 300Lys Leu Thr Lys Arg Ile Pro Leu Arg Arg Gln Val Thr Val Ser Ala305 310 315 320Glu Ser Ser Ser Ser Met Asn Ser Asn Thr Pro Leu Val Arg Ile Thr 325 330 335Thr Arg Leu Ser Ser Thr Ala Asp Thr Pro Met Leu Ala Gly Val Ser 340 345 350Glu Tyr Glu Leu Pro Glu Asp Pro Lys Trp Glu Phe Pro Arg Asp Lys 355 360 365Leu Thr Leu Gly Lys Pro Leu Gly Glu Gly Cys Phe Gly Gln Val Val 370 375 380Met Ala Glu Ala Val Gly Ile Asp Lys Asp Lys Pro Lys Glu Ala Val385 390 395 400Thr Val Ala Val Lys Met Leu Lys Asp Asp Ala Thr Glu Lys Asp Leu 405 410 415Ser Asp Leu Val Ser Glu Met Glu Met Met Lys Met Ile Gly Lys His 420 425 430Lys Asn Ile Ile Asn Leu Leu Gly Ala Cys Thr Gln Asp Gly Pro Leu 435 440 445Tyr Val Ile Val Glu Tyr Ala Ser Lys Gly Asn Leu Arg Glu Tyr Leu 450 455 460Arg Ala Arg Arg Pro Pro Gly Met Glu Tyr Ser Tyr Asp Ile Asn Arg465 470 475 480Val Pro Glu Glu Gln Met Thr Phe Lys Asp Leu Val Ser Cys Thr Tyr 485 490 495Gln Leu Ala Arg Gly Met Glu Tyr Leu Ala Ser Gln Lys Cys Ile His 500 505 510Arg Asp Leu Ala Ala Arg Asn Val Leu Val Thr Glu Asn Asn Val Met 515 520 525Lys Ile Ala Asp Phe Gly Leu Ala Arg Asp Ile Asn Asn Ile Asp Tyr 530 535 540Tyr Lys Lys Thr Thr Asn Gly Arg Leu Pro Val Lys Trp Met Ala Pro545 550 555 560Glu Ala Leu Phe Asp Arg Val Tyr Thr His Gln Ser Asp Val Trp Ser 565 570 575Phe Gly Val Leu Met Trp Glu Ile Phe Thr Leu Gly Gly Ser Pro Tyr 580 585 590Pro Gly Ile Pro Val Glu Glu Leu Phe Lys Leu Leu Lys Glu Gly His 595 600 605Arg Met Asp Lys Pro Ala Asn Cys Thr Asn Glu Leu Tyr Met Met Met 610 615 620Arg Asp Cys Trp His Ala Val Pro Ser Gln Arg Pro Thr Phe Lys Gln625 630 635 640Leu Val Glu Asp Leu Asp Arg Ile Leu Thr Leu Thr Thr Asn Glu Glu 645 650 655Tyr Leu Asp Leu Ser Gln Pro Leu Glu Gln Tyr Ser Pro Ser Tyr Pro 660 665 670Asp Thr Arg Ser Ser Cys Ser Ser Gly Asp Asp Ser Val Phe Ser Pro 675 680 685Asp Pro Met Pro Tyr Glu Pro Cys Leu Pro Gln Tyr Pro His Ile Asn 690 695 700Gly Ser Val Lys Thr70563707PRTHomo sapiens 63Met Val Ser Trp Gly Arg Phe Ile Cys Leu Val Val Val Thr Met Ala1 5 10 15Thr Leu Ser Leu Ala Arg Pro Ser Phe Ser Leu Val Glu Asp Thr Thr 20 25 30Leu Glu Pro Glu Asp Ala Ile Ser Ser Gly Asp Asp Glu Asp Asp Thr 35 40 45Asp Gly Ala Glu Asp Phe Val Ser Glu Asn Ser Asn Asn Lys Arg Ala 50 55 60Pro Tyr Trp Thr Asn Thr Glu Lys Met Glu Lys Arg Leu His Ala Val65 70 75 80Pro Ala Ala Asn Thr Val Lys Phe Arg Cys Pro Ala Gly Gly Asn Pro 85 90 95Met Pro Thr Met Arg Trp Leu Lys Asn Gly Lys Glu Phe Lys Gln Glu 100 105 110His Arg Ile Gly Gly Tyr Lys Val Arg Asn Gln His Trp Ser Leu Ile 115 120 125Met Glu Ser Val Val Pro Ser Asp Lys Gly Asn Tyr Thr Cys Val Val 130 135 140Glu Asn Glu Tyr Gly Ser Ile Asn His Thr Tyr His Leu Asp Val Val145 150 155 160Glu Arg Ser Pro His Arg Pro Ile Leu Gln Ala Gly Leu Pro Ala Asn 165 170 175Ala Ser Thr Val Val Gly Gly Asp Val Glu Phe Val Cys Lys Val Tyr 180 185 190Ser Asp Ala Gln Pro His Ile Gln Trp Ile Lys His Val Glu Lys Asn 195 200 205Gly Ser Lys Tyr Gly Pro Asp Gly Leu Pro Tyr Leu Lys Val Leu Lys 210 215 220Ala Ala Gly Val Asn Thr Thr Asp Lys Glu Ile Glu Val Leu Tyr Ile225 230 235 240Arg Asn Val Thr Phe Glu Asp Ala Gly Glu Tyr Thr Cys Leu Ala Gly 245 250 255Asn Ser Ile Gly Ile Ser Phe His Ser Ala Trp Leu Thr Val Leu Pro 260 265 270Ala Pro Gly Arg Glu Lys Glu Ile Thr Ala Ser Pro Asp Tyr Leu Glu 275 280 285Ile Ala Ile Tyr Cys Ile Gly Val Phe Leu Ile Ala Cys Met Val Val 290 295 300Thr Val Ile Leu Cys Arg Met Lys Asn Thr Thr Lys Lys Pro Asp Phe305 310 315 320Ser Ser Gln Pro Ala Val His Lys Leu Thr Lys Arg Ile Pro Leu Arg 325 330 335Arg Gln Val Thr Val Ser Ala Glu Ser Ser Ser Ser Met Asn Ser Asn 340 345 350Thr Pro Leu Val Arg Ile Thr Thr Arg Leu Ser Ser Thr Ala Asp Thr 355 360 365Pro Met Leu Ala Gly Val Ser Glu Tyr Glu Leu Pro Glu Asp Pro Lys 370 375 380Trp Glu Phe Pro Arg Asp Lys Leu Thr Leu Gly Lys Pro Leu Gly Glu385 390 395 400Gly Cys Phe Gly Gln Val Val Met Ala Glu Ala Val Gly Ile Asp Lys 405 410 415Asp Lys Pro Lys Glu Ala Val Thr Val Ala Val Lys Met Leu Lys Asp 420 425 430Asp Ala Thr Glu Lys Asp Leu Ser Asp Leu Val Ser Glu Met Glu Met 435 440 445Met Lys Met Ile Gly Lys His Lys Asn Ile Ile Asn Leu Leu Gly Ala 450 455 460Cys Thr Gln Asp Gly Pro Leu Tyr Val Ile Val Glu Tyr Ala Ser Lys465 470 475 480Gly Asn Leu Arg Glu Tyr Leu Arg Ala Arg Arg Pro Pro Gly Met Glu 485 490 495Tyr Ser Tyr Asp Ile Asn Arg Val Pro Glu Glu Gln Met Thr Phe Lys 500 505 510Asp Leu Val Ser Cys Thr Tyr Gln Leu Ala Arg Gly Met Glu Tyr Leu 515 520 525Ala Ser Gln Lys Cys Ile His Arg Asp Leu Ala Ala Arg Asn Val Leu 530 535 540Val Thr Glu Asn Asn Val Met Lys Ile Ala Asp Phe Gly Leu Ala Arg545 550 555 560Asp Ile Asn Asn Ile Asp Tyr Tyr Lys Lys Thr Thr Asn Gly Arg Leu 565 570 575Pro Val Lys Trp Met Ala Pro Glu Ala Leu Phe Asp Arg Val Tyr Thr 580 585 590His Gln Ser Asp Val Trp Ser Phe Gly Val Leu Met Trp Glu Ile Phe 595 600 605Thr Leu Gly Gly Ser Pro Tyr Pro Gly Ile Pro Val Glu Glu Leu Phe 610 615 620Lys Leu Leu Lys Glu Gly His Arg Met Asp Lys Pro Ala Asn Cys Thr625 630 635

640Asn Glu Leu Tyr Met Met Met Arg Asp Cys Trp His Ala Val Pro Ser 645 650 655Gln Arg Pro Thr Phe Lys Gln Leu Val Glu Asp Leu Asp Arg Ile Leu 660 665 670Thr Leu Thr Thr Asn Glu Glu Glu Lys Lys Val Ser Gly Ala Val Asp 675 680 685Cys His Lys Pro Pro Cys Asn Pro Ser His Leu Pro Cys Val Leu Ala 690 695 700Val Asp Gln70564706PRTHomo sapiens 64Met Val Ser Trp Gly Arg Phe Ile Cys Leu Val Val Val Thr Met Ala1 5 10 15Thr Leu Ser Leu Ala Arg Pro Ser Phe Ser Leu Val Glu Asp Thr Thr 20 25 30Leu Glu Pro Glu Gly Ala Pro Tyr Trp Thr Asn Thr Glu Lys Met Glu 35 40 45Lys Arg Leu His Ala Val Pro Ala Ala Asn Thr Val Lys Phe Arg Cys 50 55 60Pro Ala Gly Gly Asn Pro Met Pro Thr Met Arg Trp Leu Lys Asn Gly65 70 75 80Lys Glu Phe Lys Gln Glu His Arg Ile Gly Gly Tyr Lys Val Arg Asn 85 90 95Gln His Trp Ser Leu Ile Met Glu Ser Val Val Pro Ser Asp Lys Gly 100 105 110Asn Tyr Thr Cys Val Val Glu Asn Glu Tyr Gly Ser Ile Asn His Thr 115 120 125Tyr His Leu Asp Val Val Glu Arg Ser Pro His Arg Pro Ile Leu Gln 130 135 140Ala Gly Leu Pro Ala Asn Ala Ser Thr Val Val Gly Gly Asp Val Glu145 150 155 160Phe Val Cys Lys Val Tyr Ser Asp Ala Gln Pro His Ile Gln Trp Ile 165 170 175Lys His Val Glu Lys Asn Gly Ser Lys Tyr Gly Pro Asp Gly Leu Pro 180 185 190Tyr Leu Lys Val Leu Lys Ala Ala Gly Val Asn Thr Thr Asp Lys Glu 195 200 205Ile Glu Val Leu Tyr Ile Arg Asn Val Thr Phe Glu Asp Ala Gly Glu 210 215 220Tyr Thr Cys Leu Ala Gly Asn Ser Ile Gly Ile Ser Phe His Ser Ala225 230 235 240Trp Leu Thr Val Leu Pro Ala Pro Gly Arg Glu Lys Glu Ile Thr Ala 245 250 255Ser Pro Asp Tyr Leu Glu Ile Ala Ile Tyr Cys Ile Gly Val Phe Leu 260 265 270Ile Ala Cys Met Val Val Thr Val Ile Leu Cys Arg Met Lys Asn Thr 275 280 285Thr Lys Lys Pro Asp Phe Ser Ser Gln Pro Ala Val His Lys Leu Thr 290 295 300Lys Arg Ile Pro Leu Arg Arg Gln Val Thr Val Ser Ala Glu Ser Ser305 310 315 320Ser Ser Met Asn Ser Asn Thr Pro Leu Val Arg Ile Thr Thr Arg Leu 325 330 335Ser Ser Thr Ala Asp Thr Pro Met Leu Ala Gly Val Ser Glu Tyr Glu 340 345 350Leu Pro Glu Asp Pro Lys Trp Glu Phe Pro Arg Asp Lys Leu Thr Leu 355 360 365Gly Lys Pro Leu Gly Glu Gly Cys Phe Gly Gln Val Val Met Ala Glu 370 375 380Ala Val Gly Ile Asp Lys Asp Lys Pro Lys Glu Ala Val Thr Val Ala385 390 395 400Val Lys Met Leu Lys Asp Asp Ala Thr Glu Lys Asp Leu Ser Asp Leu 405 410 415Val Ser Glu Met Glu Met Met Lys Met Ile Gly Lys His Lys Asn Ile 420 425 430Ile Asn Leu Leu Gly Ala Cys Thr Gln Asp Gly Pro Leu Tyr Val Ile 435 440 445Val Glu Tyr Ala Ser Lys Gly Asn Leu Arg Glu Tyr Leu Arg Ala Arg 450 455 460Arg Pro Pro Gly Met Glu Tyr Ser Tyr Asp Ile Asn Arg Val Pro Glu465 470 475 480Glu Gln Met Thr Phe Lys Asp Leu Val Ser Cys Thr Tyr Gln Leu Ala 485 490 495Arg Gly Met Glu Tyr Leu Ala Ser Gln Lys Cys Ile His Arg Asp Leu 500 505 510Ala Ala Arg Asn Val Leu Val Thr Glu Asn Asn Val Met Lys Ile Ala 515 520 525Asp Phe Gly Leu Ala Arg Asp Ile Asn Asn Ile Asp Tyr Tyr Lys Lys 530 535 540Thr Thr Asn Gly Arg Leu Pro Val Lys Trp Met Ala Pro Glu Ala Leu545 550 555 560Phe Asp Arg Val Tyr Thr His Gln Ser Asp Val Trp Ser Phe Gly Val 565 570 575Leu Met Trp Glu Ile Phe Thr Leu Gly Gly Ser Pro Tyr Pro Gly Ile 580 585 590Pro Val Glu Glu Leu Phe Lys Leu Leu Lys Glu Gly His Arg Met Asp 595 600 605Lys Pro Ala Asn Cys Thr Asn Glu Leu Tyr Met Met Met Arg Asp Cys 610 615 620Trp His Ala Val Pro Ser Gln Arg Pro Thr Phe Lys Gln Leu Val Glu625 630 635 640Asp Leu Asp Arg Ile Leu Thr Leu Thr Thr Asn Glu Glu Tyr Leu Asp 645 650 655Leu Ser Gln Pro Leu Glu Gln Tyr Ser Pro Ser Tyr Pro Asp Thr Arg 660 665 670Ser Ser Cys Ser Ser Gly Asp Asp Ser Val Phe Ser Pro Asp Pro Met 675 680 685Pro Tyr Glu Pro Cys Leu Pro Gln Tyr Pro His Ile Asn Gly Ser Val 690 695 700Lys Thr70565705PRTHomo sapiens 65Met Val Ser Trp Gly Arg Phe Ile Cys Leu Val Val Val Thr Met Ala1 5 10 15Thr Leu Ser Leu Ala Arg Pro Ser Phe Ser Leu Val Glu Asp Thr Thr 20 25 30Leu Glu Pro Glu Glu Pro Pro Thr Lys Tyr Gln Ile Ser Gln Pro Glu 35 40 45Val Tyr Val Ala Ala Pro Gly Glu Ser Leu Glu Val Arg Cys Leu Leu 50 55 60Lys Asp Ala Ala Val Ile Ser Trp Thr Lys Asp Gly Val His Leu Gly65 70 75 80Pro Asn Asn Arg Thr Val Leu Ile Gly Glu Tyr Leu Gln Ile Lys Gly 85 90 95Ala Thr Pro Arg Asp Ser Gly Leu Tyr Ala Cys Thr Ala Ser Arg Thr 100 105 110Val Asp Ser Glu Thr Trp Tyr Phe Met Val Asn Val Thr Asp Ala Ile 115 120 125Ser Ser Gly Asp Asp Glu Asp Asp Thr Asp Gly Ala Glu Asp Phe Val 130 135 140Ser Glu Asn Ser Asn Asn Lys Arg Ala Pro Tyr Trp Thr Asn Thr Glu145 150 155 160Lys Met Glu Lys Arg Leu His Ala Val Pro Ala Ala Asn Thr Val Lys 165 170 175Phe Arg Cys Pro Ala Gly Gly Asn Pro Met Pro Thr Met Arg Trp Leu 180 185 190Lys Asn Gly Lys Glu Phe Lys Gln Glu His Arg Ile Gly Gly Tyr Lys 195 200 205Val Arg Asn Gln His Trp Ser Leu Ile Met Glu Ser Val Val Pro Ser 210 215 220Asp Lys Gly Asn Tyr Thr Cys Val Val Glu Asn Glu Tyr Gly Ser Ile225 230 235 240Asn His Thr Tyr His Leu Asp Val Val Glu Arg Ser Pro His Arg Pro 245 250 255Ile Leu Gln Ala Gly Leu Pro Ala Asn Ala Ser Thr Val Val Gly Gly 260 265 270Asp Val Glu Phe Val Cys Lys Val Tyr Ser Asp Ala Gln Pro His Ile 275 280 285Gln Trp Ile Lys His Val Glu Lys Asn Gly Ser Lys Tyr Gly Pro Asp 290 295 300Gly Leu Pro Tyr Leu Lys Val Leu Lys Val Ser Ala Glu Ser Ser Ser305 310 315 320Ser Met Asn Ser Asn Thr Pro Leu Val Arg Ile Thr Thr Arg Leu Ser 325 330 335Ser Thr Ala Asp Thr Pro Met Leu Ala Gly Val Ser Glu Tyr Glu Leu 340 345 350Pro Glu Asp Pro Lys Trp Glu Phe Pro Arg Asp Lys Leu Thr Leu Gly 355 360 365Lys Pro Leu Gly Glu Gly Cys Phe Gly Gln Val Val Met Ala Glu Ala 370 375 380Val Gly Ile Asp Lys Asp Lys Pro Lys Glu Ala Val Thr Val Ala Val385 390 395 400Lys Met Leu Lys Asp Asp Ala Thr Glu Lys Asp Leu Ser Asp Leu Val 405 410 415Ser Glu Met Glu Met Met Lys Met Ile Gly Lys His Lys Asn Ile Ile 420 425 430Asn Leu Leu Gly Ala Cys Thr Gln Asp Gly Pro Leu Tyr Val Ile Val 435 440 445Glu Tyr Ala Ser Lys Gly Asn Leu Arg Glu Tyr Leu Arg Ala Arg Arg 450 455 460Pro Pro Gly Met Glu Tyr Ser Tyr Asp Ile Asn Arg Val Pro Glu Glu465 470 475 480Gln Met Thr Phe Lys Asp Leu Val Ser Cys Thr Tyr Gln Leu Ala Arg 485 490 495Gly Met Glu Tyr Leu Ala Ser Gln Lys Cys Ile His Arg Asp Leu Ala 500 505 510Ala Arg Asn Val Leu Val Thr Glu Asn Asn Val Met Lys Ile Ala Asp 515 520 525Phe Gly Leu Ala Arg Asp Ile Asn Asn Ile Asp Tyr Tyr Lys Lys Thr 530 535 540Thr Asn Gly Arg Leu Pro Val Lys Trp Met Ala Pro Glu Ala Leu Phe545 550 555 560Asp Arg Val Tyr Thr His Gln Ser Asp Val Trp Ser Phe Gly Val Leu 565 570 575Met Trp Glu Ile Phe Thr Leu Gly Gly Ser Pro Tyr Pro Gly Ile Pro 580 585 590Val Glu Glu Leu Phe Lys Leu Leu Lys Glu Gly His Arg Met Asp Lys 595 600 605Pro Ala Asn Cys Thr Asn Glu Leu Tyr Met Met Met Arg Asp Cys Trp 610 615 620His Ala Val Pro Ser Gln Arg Pro Thr Phe Lys Gln Leu Val Glu Asp625 630 635 640Leu Asp Arg Ile Leu Thr Leu Thr Thr Asn Glu Glu Tyr Leu Asp Leu 645 650 655Ser Gln Pro Leu Glu Gln Tyr Ser Pro Ser Tyr Pro Asp Thr Arg Ser 660 665 670Ser Cys Ser Ser Gly Asp Asp Ser Val Phe Ser Pro Asp Pro Met Pro 675 680 685Tyr Glu Pro Cys Leu Pro Gln Tyr Pro His Ile Asn Gly Ser Val Lys 690 695 700Thr70566704PRTHomo sapiens 66Met Val Ser Trp Gly Arg Phe Ile Cys Leu Val Val Val Thr Met Ala1 5 10 15Thr Leu Ser Leu Ala Arg Pro Ser Phe Ser Leu Val Glu Asp Thr Thr 20 25 30Leu Glu Pro Glu Gly Ala Pro Tyr Trp Thr Asn Thr Glu Lys Met Glu 35 40 45Lys Arg Leu His Ala Val Pro Ala Ala Asn Thr Val Lys Phe Arg Cys 50 55 60Pro Ala Gly Gly Asn Pro Met Pro Thr Met Arg Trp Leu Lys Asn Gly65 70 75 80Lys Glu Phe Lys Gln Glu His Arg Ile Gly Gly Tyr Lys Val Arg Asn 85 90 95Gln His Trp Ser Leu Ile Met Glu Ser Val Val Pro Ser Asp Lys Gly 100 105 110Asn Tyr Thr Cys Val Val Glu Asn Glu Tyr Gly Ser Ile Asn His Thr 115 120 125Tyr His Leu Asp Val Val Glu Arg Ser Pro His Arg Pro Ile Leu Gln 130 135 140Ala Gly Leu Pro Ala Asn Ala Ser Thr Val Val Gly Gly Asp Val Glu145 150 155 160Phe Val Cys Lys Val Tyr Ser Asp Ala Gln Pro His Ile Gln Trp Ile 165 170 175Lys His Val Glu Lys Asn Gly Ser Lys Tyr Gly Pro Asp Gly Leu Pro 180 185 190Tyr Leu Lys Val Leu Lys Ala Ala Gly Val Asn Thr Thr Asp Lys Glu 195 200 205Ile Glu Val Leu Tyr Ile Arg Asn Val Thr Phe Glu Asp Ala Gly Glu 210 215 220Tyr Thr Cys Leu Ala Gly Asn Ser Ile Gly Ile Ser Phe His Ser Ala225 230 235 240Trp Leu Thr Val Leu Pro Ala Pro Gly Arg Glu Lys Glu Ile Thr Ala 245 250 255Ser Pro Asp Tyr Leu Glu Ile Ala Ile Tyr Cys Ile Gly Val Phe Leu 260 265 270Ile Ala Cys Met Val Val Thr Val Ile Leu Cys Arg Met Lys Asn Thr 275 280 285Thr Lys Lys Pro Asp Phe Ser Ser Gln Pro Ala Val His Lys Leu Thr 290 295 300Lys Arg Ile Pro Leu Arg Arg Gln Val Ser Ala Glu Ser Ser Ser Ser305 310 315 320Met Asn Ser Asn Thr Pro Leu Val Arg Ile Thr Thr Arg Leu Ser Ser 325 330 335Thr Ala Asp Thr Pro Met Leu Ala Gly Val Ser Glu Tyr Glu Leu Pro 340 345 350Glu Asp Pro Lys Trp Glu Phe Pro Arg Asp Lys Leu Thr Leu Gly Lys 355 360 365Pro Leu Gly Glu Gly Cys Phe Gly Gln Val Val Met Ala Glu Ala Val 370 375 380Gly Ile Asp Lys Asp Lys Pro Lys Glu Ala Val Thr Val Ala Val Lys385 390 395 400Met Leu Lys Asp Asp Ala Thr Glu Lys Asp Leu Ser Asp Leu Val Ser 405 410 415Glu Met Glu Met Met Lys Met Ile Gly Lys His Lys Asn Ile Ile Asn 420 425 430Leu Leu Gly Ala Cys Thr Gln Asp Gly Pro Leu Tyr Val Ile Val Glu 435 440 445Tyr Ala Ser Lys Gly Asn Leu Arg Glu Tyr Leu Arg Ala Arg Arg Pro 450 455 460Pro Gly Met Glu Tyr Ser Tyr Asp Ile Asn Arg Val Pro Glu Glu Gln465 470 475 480Met Thr Phe Lys Asp Leu Val Ser Cys Thr Tyr Gln Leu Ala Arg Gly 485 490 495Met Glu Tyr Leu Ala Ser Gln Lys Cys Ile His Arg Asp Leu Ala Ala 500 505 510Arg Asn Val Leu Val Thr Glu Asn Asn Val Met Lys Ile Ala Asp Phe 515 520 525Gly Leu Ala Arg Asp Ile Asn Asn Ile Asp Tyr Tyr Lys Lys Thr Thr 530 535 540Asn Gly Arg Leu Pro Val Lys Trp Met Ala Pro Glu Ala Leu Phe Asp545 550 555 560Arg Val Tyr Thr His Gln Ser Asp Val Trp Ser Phe Gly Val Leu Met 565 570 575Trp Glu Ile Phe Thr Leu Gly Gly Ser Pro Tyr Pro Gly Ile Pro Val 580 585 590Glu Glu Leu Phe Lys Leu Leu Lys Glu Gly His Arg Met Asp Lys Pro 595 600 605Ala Asn Cys Thr Asn Glu Leu Tyr Met Met Met Arg Asp Cys Trp His 610 615 620Ala Val Pro Ser Gln Arg Pro Thr Phe Lys Gln Leu Val Glu Asp Leu625 630 635 640Asp Arg Ile Leu Thr Leu Thr Thr Asn Glu Glu Tyr Leu Asp Leu Ser 645 650 655Gln Pro Leu Glu Gln Tyr Ser Pro Ser Tyr Pro Asp Thr Arg Ser Ser 660 665 670Cys Ser Ser Gly Asp Asp Ser Val Phe Ser Pro Asp Pro Met Pro Tyr 675 680 685Glu Pro Cys Leu Pro Gln Tyr Pro His Ile Asn Gly Ser Val Lys Thr 690 695 70067680PRTHomo sapiens 67Met Val Ser Trp Gly Arg Phe Ile Cys Leu Val Val Val Thr Met Ala1 5 10 15Thr Leu Ser Leu Ala Arg Pro Ser Phe Ser Leu Val Glu Asp Thr Thr 20 25 30Leu Glu Pro Glu Asp Ala Ile Ser Ser Gly Asp Asp Glu Asp Asp Thr 35 40 45Asp Gly Ala Glu Asp Phe Val Ser Glu Asn Ser Asn Asn Lys Arg Ala 50 55 60Pro Tyr Trp Thr Asn Thr Glu Lys Met Glu Lys Arg Leu His Ala Val65 70 75 80Pro Ala Ala Asn Thr Val Lys Phe Arg Cys Pro Ala Gly Gly Asn Pro 85 90 95Met Pro Thr Met Arg Trp Leu Lys Asn Gly Lys Glu Phe Lys Gln Glu 100 105 110His Arg Ile Gly Gly Tyr Lys Val Arg Asn Gln His Trp Ser Leu Ile 115 120 125Met Glu Ser Val Val Pro Ser Asp Lys Gly Asn Tyr Thr Cys Val Val 130 135 140Glu Asn Glu Tyr Gly Ser Ile Asn His Thr Tyr His Leu Asp Val Val145 150 155 160Glu Arg Ser Pro His Arg Pro Ile Leu Gln Ala Gly Leu Pro Ala Asn 165 170 175Ala Ser Thr Val Val Gly Gly Asp Val Glu Phe Val Cys Lys Val Tyr 180 185 190Ser Asp Ala Gln Pro His Ile Gln Trp Ile Lys His Val Glu Lys Asn 195 200 205Gly Ser Lys Tyr Gly Pro Asp Gly Leu Pro Tyr Leu Lys Val Leu Lys 210 215 220His Ser Gly Ile Asn Ser Ser Asn Ala Glu Val Leu Ala Leu Phe Asn225 230 235 240Val Thr Glu Ala Asp Ala Gly Glu Tyr Ile Cys Lys Val Ser Asn Tyr 245 250 255Ile Gly Gln Ala Asn Gln Ser Ala Trp Leu Thr Val Leu Pro Lys Gln 260 265 270Gln Ala Pro Gly Arg Glu Lys Glu Ile Thr Ala Ser Pro Asp Tyr Leu 275

280 285Glu Ile Ala Ile Tyr Cys Ile Gly Val Phe Leu Ile Ala Cys Met Val 290 295 300Val Thr Val Ile Leu Cys Arg Met Lys Asn Thr Thr Lys Lys Pro Asp305 310 315 320Phe Ser Ser Gln Pro Ala Val His Lys Leu Thr Lys Arg Ile Pro Leu 325 330 335Arg Arg Gln Val Thr Val Ser Ala Glu Ser Ser Ser Ser Met Asn Ser 340 345 350Asn Thr Pro Leu Val Arg Ile Thr Thr Arg Leu Ser Ser Thr Ala Asp 355 360 365Thr Pro Met Leu Ala Gly Val Ser Glu Tyr Glu Leu Pro Glu Asp Pro 370 375 380Lys Trp Glu Phe Pro Arg Asp Lys Leu Thr Leu Gly Lys Pro Leu Gly385 390 395 400Glu Gly Cys Phe Gly Gln Val Val Met Ala Glu Ala Val Gly Ile Asp 405 410 415Lys Asp Lys Pro Lys Glu Ala Val Thr Val Ala Val Lys Met Leu Lys 420 425 430Asp Asp Ala Thr Glu Lys Asp Leu Ser Asp Leu Val Ser Glu Met Glu 435 440 445Met Met Lys Met Ile Gly Lys His Lys Asn Ile Ile Asn Leu Leu Gly 450 455 460Ala Cys Thr Gln Asp Gly Pro Leu Tyr Val Ile Val Glu Tyr Ala Ser465 470 475 480Lys Gly Asn Leu Arg Glu Tyr Leu Arg Ala Arg Arg Pro Pro Gly Met 485 490 495Glu Tyr Ser Tyr Asp Ile Asn Arg Val Pro Glu Glu Gln Met Thr Phe 500 505 510Lys Asp Leu Val Ser Cys Thr Tyr Gln Leu Ala Arg Gly Met Glu Tyr 515 520 525Leu Ala Ser Gln Lys Cys Ile His Arg Asp Leu Ala Ala Arg Asn Val 530 535 540Leu Val Thr Glu Asn Asn Val Met Lys Ile Ala Asp Phe Gly Leu Ala545 550 555 560Arg Asp Ile Asn Asn Ile Asp Tyr Tyr Lys Lys Thr Thr Asn Gly Arg 565 570 575Leu Pro Val Lys Trp Met Ala Pro Glu Ala Leu Phe Asp Arg Val Tyr 580 585 590Thr His Gln Ser Asp Val Trp Ser Phe Gly Val Leu Met Trp Glu Ile 595 600 605Phe Thr Leu Gly Gly Ser Pro Tyr Pro Gly Ile Pro Val Glu Glu Leu 610 615 620Phe Lys Leu Leu Lys Glu Gly His Arg Met Asp Lys Pro Ala Asn Cys625 630 635 640Thr Asn Glu Leu Tyr Met Met Met Arg Asp Cys Trp His Ala Val Pro 645 650 655Ser Gln Arg Pro Thr Phe Lys Gln Leu Val Glu Asp Leu Asp Arg Ile 660 665 670Leu Thr Leu Thr Thr Asn Glu Ile 675 68068396PRTHomo sapiens 68Met Val Ser Trp Gly Arg Phe Ile Cys Leu Val Val Val Thr Met Ala1 5 10 15Thr Leu Ser Leu Ala Arg Pro Ser Phe Ser Leu Val Glu Asp Thr Thr 20 25 30Leu Glu Pro Glu Glu Pro Pro Thr Lys Tyr Gln Ile Ser Gln Pro Glu 35 40 45Val Tyr Val Ala Ala Pro Gly Glu Ser Leu Glu Val Arg Cys Leu Leu 50 55 60Lys Asp Ala Ala Val Ile Ser Trp Thr Lys Asp Gly Val His Leu Gly65 70 75 80Pro Asn Asn Arg Thr Val Leu Ile Gly Glu Tyr Leu Gln Ile Lys Gly 85 90 95Ala Thr Pro Arg Asp Ser Gly Leu Tyr Ala Cys Thr Ala Ser Arg Thr 100 105 110Val Asp Ser Glu Thr Trp Tyr Phe Met Val Asn Val Thr Asp Ala Ile 115 120 125Ser Ser Gly Asp Asp Glu Asp Asp Thr Asp Gly Ala Glu Asp Phe Val 130 135 140Ser Glu Asn Ser Asn Asn Lys Arg Ala Pro Tyr Trp Thr Asn Thr Glu145 150 155 160Lys Thr Glu Lys Arg Leu His Ala Val Pro Ala Ala Asn Thr Val Lys 165 170 175Phe Arg Cys Pro Ala Gly Gly Asn Pro Met Pro Thr Met Arg Trp Leu 180 185 190Lys Asn Gly Lys Glu Phe Lys Gln Glu His Arg Ile Gly Gly Tyr Lys 195 200 205Val Arg Asn Gln His Trp Ser Leu Ile Met Glu Ser Val Val Pro Ser 210 215 220Asp Lys Gly Asn Tyr Thr Cys Val Val Glu Asn Glu Tyr Gly Ser Ile225 230 235 240Asn His Thr Tyr His Leu Asp Val Val Glu Arg Ser Pro His Arg Pro 245 250 255Ile Leu Gln Ala Gly Leu Pro Ala Asn Ala Ser Thr Val Val Gly Gly 260 265 270Asp Val Glu Phe Val Cys Lys Val Tyr Ser Asp Ala Gln Pro His Ile 275 280 285Gln Trp Ile Lys His Val Glu Lys Asn Gly Ser Lys Tyr Gly Pro Asp 290 295 300Gly Leu Pro Tyr Leu Lys Val Leu Lys Ala Ala Gly Val Asn Thr Thr305 310 315 320Asp Lys Glu Ile Glu Val Leu Tyr Ile Arg Asn Val Thr Phe Glu Asp 325 330 335Ala Gly Glu Tyr Thr Cys Leu Ala Gly Asn Ser Ile Gly Ile Ser Phe 340 345 350His Ser Ala Trp Leu Thr Val Leu Pro Gly Ile Tyr Cys Ser Phe Ser 355 360 365Leu Gly Phe Phe Pro Phe Ser Trp Leu Thr Ala Ile Lys Leu Thr Gln 370 375 380Leu Leu Leu Ser Glu Met Ala Pro Phe Ile Leu Ala385 390 39569317PRTHomo sapiens 69Met Val Ser Trp Gly Arg Phe Ile Cys Leu Val Val Val Thr Met Ala1 5 10 15Thr Leu Ser Leu Ala Arg Pro Ser Phe Ser Leu Val Glu Asp Thr Thr 20 25 30Leu Glu Pro Glu Glu Pro Pro Thr Lys Tyr Gln Ile Ser Gln Pro Glu 35 40 45Val Tyr Val Ala Ala Pro Gly Glu Ser Leu Glu Val Arg Cys Leu Leu 50 55 60Lys Asp Ala Ala Val Ile Ser Trp Thr Lys Asp Gly Val His Leu Gly65 70 75 80Pro Asn Asn Arg Thr Val Leu Ile Gly Glu Tyr Leu Gln Ile Lys Gly 85 90 95Ala Thr Pro Arg Asp Ser Gly Leu Tyr Ala Cys Thr Ala Ser Arg Thr 100 105 110Val Asp Ser Glu Thr Trp Tyr Phe Met Val Asn Val Thr Asp Ala Ile 115 120 125Ser Ser Gly Asp Asp Glu Asp Asp Thr Asp Gly Ala Glu Asp Phe Val 130 135 140Ser Glu Asn Ser Asn Asn Lys Arg Ala Pro Tyr Trp Thr Asn Thr Glu145 150 155 160Lys Met Glu Lys Arg Leu His Ala Val Pro Ala Ala Asn Thr Val Lys 165 170 175Phe Arg Cys Pro Ala Gly Gly Asn Pro Met Pro Thr Met Arg Trp Leu 180 185 190Lys Asn Gly Lys Glu Phe Lys Gln Glu His Arg Ile Gly Gly Tyr Lys 195 200 205Val Arg Asn Gln His Trp Ser Leu Ile Met Glu Ser Val Val Pro Ser 210 215 220Asp Lys Gly Asn Tyr Thr Cys Val Val Glu Asn Glu Tyr Gly Ser Ile225 230 235 240Asn His Thr Tyr His Leu Asp Val Val Glu Arg Ser Pro His Arg Pro 245 250 255Ile Leu Gln Ala Gly Leu Pro Ala Asn Ala Ser Thr Val Val Gly Gly 260 265 270Asp Val Glu Phe Val Cys Lys Val Tyr Ser Asp Ala Gln Pro His Ile 275 280 285Gln Trp Ile Lys His Val Glu Lys Asn Gly Ser Lys Tyr Gly Pro Asp 290 295 300Gly Leu Pro Tyr Leu Lys Val Leu Lys Val Arg Thr Phe305 310 31570266PRTHomo sapiens 70Met Val Ser Trp Gly Arg Phe Ile Cys Leu Val Val Val Thr Met Ala1 5 10 15Thr Leu Ser Leu Ala Arg Pro Ser Phe Ser Leu Val Glu Asp Thr Thr 20 25 30Leu Glu Pro Glu Glu Pro Pro Thr Lys Tyr Gln Ile Ser Gln Pro Glu 35 40 45Val Tyr Val Ala Ala Pro Gly Glu Ser Leu Glu Val Arg Cys Leu Leu 50 55 60Lys Asp Ala Ala Val Ile Ser Trp Thr Lys Asp Gly Val His Leu Gly65 70 75 80Pro Asn Asn Arg Thr Val Leu Ile Gly Glu Tyr Leu Gln Ile Lys Gly 85 90 95Ala Thr Pro Arg Asp Ser Gly Leu Tyr Ala Cys Thr Ala Ser Arg Thr 100 105 110Val Asp Ser Glu Thr Trp Tyr Phe Met Val Asn Val Thr Asp Ala Ile 115 120 125Ser Ser Gly Asp Asp Glu Asp Asp Thr Asp Gly Ala Glu Asp Phe Val 130 135 140Ser Glu Asn Ser Asn Asn Lys Arg Ala Pro Tyr Trp Thr Asn Thr Glu145 150 155 160Lys Met Glu Lys Arg Leu His Ala Val Pro Ala Ala Asn Thr Val Lys 165 170 175Phe Arg Cys Pro Ala Gly Gly Asn Pro Met Pro Thr Met Arg Trp Leu 180 185 190Lys Asn Gly Lys Glu Phe Lys Gln Glu His Arg Ile Gly Gly Tyr Lys 195 200 205Val Arg Asn Gln His Trp Ser Leu Ile Met Glu Ser Val Val Pro Ser 210 215 220Asp Lys Gly Asn Tyr Thr Cys Val Val Glu Asn Glu Tyr Gly Ser Ile225 230 235 240Asn His Thr Tyr His Leu Asp Val Val Gly Glu Ser Ala Ser Pro Arg 245 250 255Val Ala Ala Ala Tyr Gln Pro Ile Leu Ala 260 26571336DNAMus musculus 71caggtgaagc tgcaggagtc tggcgctgag ttggtgaaac ctggggcttc agtgaagata 60tcctgcaagg cttctggcta catcttcact gaccatgctc ttcactgggt gaggcagaag 120cctgaacagg gcctggaatg gattgggtat atttttcccg gaaatggtaa tattgagtac 180aatgagaagt tcaagggcaa ggccacactg actgcagaca aatcctccag tactgcctac 240atgcagctca acagcctgac atctggagat tctgcaatgt atttctgtaa aaagatggac 300tactggggcc aagggaccac ggtcaccgtc tcctca 33672111PRTMus musculus 72Val Lys Leu Gln Glu Ser Gly Ala Glu Leu Val Lys Pro Gly Ala Ser1 5 10 15Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Asp His Ala 20 25 30Leu His Trp Val Arg Gln Lys Pro Glu Gln Gly Leu Glu Trp Ile Gly 35 40 45Tyr Ile Phe Pro Gly Asn Gly Asn Ile Glu Tyr Asn Glu Lys Phe Lys 50 55 60Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr Met65 70 75 80Gln Leu Asn Ser Leu Thr Ser Gly Asp Ser Ala Met Tyr Phe Cys Lys 85 90 95Lys Met Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 100 105 11073336DNAMus musculus 73caggtgaagc tgcaggagtc tggcgctgag ttggtgaaac ctggggcttc agtgaagatc 60tcctgcaagg cttctggtta caccttcact gaccattcta ttcactgggt gaagcagaag 120cctggacagg gcctagaatg gattggatat ctttttcccg gaaatggtaa ttttgaatat 180aatgagaaat tcaagggcaa ggccacactg actgcagaca aatcctccag cactgcctac 240atgcacctca acagcctgac atctgaggat tctgcagtgt atttctgtaa aaagatggac 300tactggggcc aagggaccac ggtcaccgtc tcctca 33674111PRTMus musculus 74Val Lys Leu Gln Glu Ser Gly Ala Glu Leu Val Lys Pro Gly Ala Ser1 5 10 15Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp His Ser 20 25 30Ile His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile Gly 35 40 45Tyr Leu Phe Pro Gly Asn Gly Asn Phe Glu Tyr Asn Glu Lys Phe Lys 50 55 60Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr Met65 70 75 80His Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys Lys 85 90 95Lys Met Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 100 105 11075336DNAMus musculus 75caggttcagc tgcagcagtc cgacgctgag ttggtgaaac ctggggcttc agtgaagata 60tcctgcaggg cttctggcta caccttcact gaccattcta ttcactgggt gaagcagcag 120cctggccagg gcctggaatg gatcggatat atttttcccg gaaatggaaa tattgaatac 180aatgacaaat tcaagggcaa ggccacactg actgcagaca aatcctccgg cactgcctac 240atgcagctca acagcctgac atctgaggat tctgcagtgt atttctgtaa aaggatgggg 300tactggggtc aaggaacctc agtcaccgtc tcctca 33676111PRTMus musculus 76Val Gln Leu Gln Gln Ser Asp Ala Glu Leu Val Lys Pro Gly Ala Ser1 5 10 15Val Lys Ile Ser Cys Arg Ala Ser Gly Tyr Thr Phe Thr Asp His Ser 20 25 30Ile His Trp Val Lys Gln Gln Pro Gly Gln Gly Leu Glu Trp Ile Gly 35 40 45Tyr Ile Phe Pro Gly Asn Gly Asn Ile Glu Tyr Asn Asp Lys Phe Lys 50 55 60Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Gly Thr Ala Tyr Met65 70 75 80Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys Lys 85 90 95Arg Met Gly Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser 100 105 11077360DNAMus musculus 77caggtcaagc tgcaggagtc tggacctgaa ctggtaaagc ctggggcttc agtgaagatg 60tcctgcaagg cttctggata cacattcact aactatgtta tacactgggt gaagcaaaag 120cctgggcagg gccttgagtg gattggatat attaatcctt acaatgatgg ctctaagtac 180aatgagaagt tcaaaggcaa ggcctcactg acttcagaca aatcctccag cacagcctac 240atggagctca gcagcctgac ctctgaggac tctgcggtct attactgtgc aagacatctc 300gctaatacct actactactt tgactactgg ggccaaggga ccacggtcac cgtctcctca 36078119PRTMus musculus 78Val Lys Leu Gln Glu Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser1 5 10 15Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr Val 20 25 30Ile His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile Gly 35 40 45Tyr Ile Asn Pro Tyr Asn Asp Gly Ser Lys Tyr Asn Glu Lys Phe Lys 50 55 60Gly Lys Ala Ser Leu Thr Ser Asp Lys Ser Ser Ser Thr Ala Tyr Met65 70 75 80Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala 85 90 95Arg His Leu Ala Asn Thr Tyr Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110Thr Thr Val Thr Val Ser Ser 11579336DNAMus musculus 79caggtcaagc tgcaggagtc tggcgctgag ttggtgaaac ctggggcttc agtgaagatc 60tcctgcaagg cttctggcta caccttcact gaccattcta ttcactgggt gaagcagaag 120cctggacagg gcctagaatg gattggatat ctttttcccg gaaatggtaa ttttgagtac 180aatgaaaaat tcaagggcaa ggccacactg actgcagaca aatcctccag cactgtctac 240atgtacctca acagcctgac atctgaggat tctgcagtgt atttctgtaa aaggatgggg 300tactggggcc aagggaccac ggtcaccgtc tcctca 33680111PRTMus musculus 80Val Lys Leu Gln Glu Ser Gly Ala Glu Leu Val Lys Pro Gly Ala Ser1 5 10 15Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp His Ser 20 25 30Ile His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile Gly 35 40 45Tyr Leu Phe Pro Gly Asn Gly Asn Phe Glu Tyr Asn Glu Lys Phe Lys 50 55 60Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Val Tyr Met65 70 75 80Tyr Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys Lys 85 90 95Arg Met Gly Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 100 105 11081357DNAMus musculus 81gtgaagctgc aggagtctgg acctgaactg gtaaagcctg gggcttcagt gaagatgtcc 60tgcaaggctt ctggatacac attcactaac tatgttatac actgggtgaa gcaaaagcct 120gggcagggcc ttgagtggat tggatatatt aatccttaca atgatggctc taagtacaat 180gagaagttca aaggcaaggc ctcactgact tcagacaaat cctccagcac agcctacatg 240gagctcagca gcctgacctc tgaggactct gcggtctatt actgtgcaag acatctcgct 300aatacctact actactttga ctactggggc caaggcacca ctctcacagt ctcctca 35782120PRTMus musculus 82Gln Val Gln Leu Gln Glu Ser Gly Pro Glu Leu Val Lys Pro Gly Ala1 5 10 15Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30Val Ile His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Ser Lys Tyr Asn Glu Lys Phe 50 55 60Lys Gly Lys Ala Ser Leu Thr Ser Asp Lys Ser Ser Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95Ala Arg His Leu Ala Asn Thr Tyr Tyr Tyr Phe Asp Tyr Trp Gly Gln 100 105 110Gly Thr Thr Leu Thr Val Ser Ser 115 12083342DNAMus musculus 83gatgttttga tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60atctcttgca gatctagtca

gagcattgta catagtaatg gaaacaccta tttagaatgg 120tacctgcaga aaccaggcca gtctccaaag ctcctgatct acaaagtttc caaccgattt 180tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc 240agcagagtgg aggctgagga tctgggagtt tattactgct ttcaaggttc acatgttcct 300cctacgttcg gtgctgggac caagctggag ctgaaacggg ct 34284113PRTMus musculus 84Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly1 5 10 15Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95Ser His Val Pro Pro Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 110Arg85324DNAMus musculus 85gacatccaga tgactcagtc tccagcctcc ctatctgcat ctgtgggaga aactgtcacc 60atcacatgtc gaacaactga aaatatttac agttattttg tatggtctca gcagagacag 120ggaaaatctc ctcagctccg ggtctataat gcaaaatcct tagcagaagg tgtgccatca 180agtttcaatg tcagtgtatc aggcacacag ttttctctga agatcaatag cctgcagcct 240gaagattttg ggacttatca ctgtcaacac cattatggta ctccgtacac gttcggaggg 300gggaccaggc tggaaataag acgg 32486108PRTMus musculus 86Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly1 5 10 15Glu Thr Val Thr Ile Thr Cys Arg Thr Thr Glu Asn Ile Tyr Ser Tyr 20 25 30Phe Val Trp Ser Gln Gln Arg Gln Gly Lys Ser Pro Gln Leu Arg Val 35 40 45Tyr Asn Ala Lys Ser Leu Ala Glu Gly Val Pro Ser Ser Phe Asn Val 50 55 60Ser Val Ser Gly Thr Gln Phe Ser Leu Lys Ile Asn Ser Leu Gln Pro65 70 75 80Glu Asp Phe Gly Thr Tyr His Cys Gln His His Tyr Gly Thr Pro Tyr 85 90 95Thr Phe Gly Gly Gly Thr Arg Leu Glu Ile Arg Arg 100 10587336DNAMus musculus 87gacattgtgc tgacacagtc tcctgcttcc ttagctgtat ctctggggca gagggccacc 60atctcgtaca gggccagcaa aagtgtcagt acatctggct atagttatat gcactggaac 120caacagaaac caggacagcc acccagactc ctcatctatc ttgtatccaa cctagaatct 180ggggtccctg ccaggttcag tggcagtggg tctgggacag acttcaccct caacatccat 240cctgtggagg aggaggatgc tgcaacctat tactgtcagc acattaggga gcttacacgt 300tcggaggggg gcaccaagct ggaaatcaaa cggaga 33688112PRTMus musculus 88Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly1 5 10 15Gln Arg Ala Thr Ile Ser Tyr Arg Ala Ser Lys Ser Val Ser Thr Ser 20 25 30Gly Tyr Ser Tyr Met His Trp Asn Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45Arg Leu Leu Ile Tyr Leu Val Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His65 70 75 80Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ile Arg 85 90 95Glu Leu Thr Arg Ser Glu Gly Gly Thr Lys Leu Glu Ile Lys Arg Arg 100 105 11089327DNAMus musculus 89gacatcaaga tgacccagtc tccatcctcc atgtatgcat cgctgggaga gagagtcact 60atcacttgca aggcgagtca ggacattaaa agctatttaa gctggtacca gcagaaacca 120tggaaatctc ctaagaccct gatctattat gcaacaagct tggcagatgg ggtcccatca 180agattcagtg gcagtggatc tgggcaagat tattctctaa ccatcagcag cctggagtct 240gacgatacag caacttatta ctgtctacag catggtgaga gcccgtacac gttcggaggg 300gggaccaagc tggaaataaa acgggct 32790108PRTMus musculus 90Asp Ile Lys Met Thr Gln Ser Pro Ser Ser Met Tyr Ala Ser Leu Gly1 5 10 15Glu Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Lys Ser Tyr 20 25 30Leu Ser Trp Tyr Gln Gln Lys Pro Trp Lys Ser Pro Lys Thr Leu Ile 35 40 45Tyr Tyr Ala Thr Ser Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Gln Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Ser65 70 75 80Asp Asp Thr Ala Thr Tyr Tyr Cys Leu Gln His Gly Glu Ser Pro Phe 85 90 95Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg 100 10591327DNAMus musculus 91gatgttgtgc taactcagtc tcctgccacc ctgtctgtga ctccaggaga tagagtcagt 60ctttcctgca gggccagcca aaatattggc aactacctac actggtatca acagaaatca 120catgagtctc caaggcttct catcaagtat gcttcccagt ccatctctgg gatcccctcc 180aggttcagtg gcagtggatc agtcacagat ttcactctca atatcaacag tgtggagact 240gaagattttg gaatgtattt ctgtcaacag agtgacacct ggcctctcac gttcggtgct 300gggaccaagc tggagctgaa acgggct 32792108PRTMus musculus 92Asp Val Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Val Thr Pro Gly1 5 10 15Asp Arg Val Ser Leu Ser Cys Arg Ala Ser Gln Asn Ile Gly Asn Tyr 20 25 30Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile 35 40 45Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Val Thr Asp Phe Thr Leu Asn Ile Asn Ser Val Glu Thr65 70 75 80Glu Asp Phe Gly Met Tyr Phe Cys Gln Gln Ser Asp Thr Trp Pro Leu 85 90 95Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg 100 105938PRTMus musculus 93Thr Phe Thr Asp His Ser Ile His1 5948PRTMus musculus 94Thr Phe Thr Asn Tyr Val Ile His1 5958PRTMus musculus 95Ile Phe Thr Asp His Ala Leu His1 59617PRTMus musculus 96Tyr Ile Phe Pro Gly Asn Gly Asn Ile Glu Tyr Asn Asp Lys Phe Lys1 5 10 15Gly9717PRTMus musculus 97Tyr Leu Phe Pro Gly Asn Gly Asn Phe Glu Tyr Asn Glu Lys Phe Lys1 5 10 15Gly9817PRTMus musculus 98Tyr Ile Asn Pro Tyr Asn Asp Gly Ser Lys Tyr Asn Glu Lys Phe Lys1 5 10 15Gly9917PRTMus musculus 99Tyr Ile Phe Pro Gly Asn Gly Asn Ile Glu Tyr Asn Glu Lys Phe Lys1 5 10 15Gly1005PRTMus musculus 100Lys Arg Met Gly Tyr1 51015PRTMus musculus 101Lys Lys Met Asp Tyr1 510213PRTMus musculus 102Ala Arg His Leu Ala Asn Thr Tyr Tyr Tyr Phe Asp Tyr1 5 1010316PRTMus musculus 103Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr Leu Glu1 5 10 1510411PRTMus musculus 104Arg Thr Thr Glu Asn Ile Tyr Ser Tyr Phe Val1 5 1010515PRTMus musculus 105Arg Ala Ser Lys Ser Val Ser Thr Ser Gly Tyr Ser Tyr Met His1 5 10 1510611PRTMus musculus 106Lys Ala Ser Gln Asp Ile Lys Ser Tyr Leu Ser1 5 1010711PRTMus musculus 107Arg Ala Ser Gln Asn Ile Gly Asn Tyr Leu His1 5 101087PRTMus musculus 108Lys Val Ser Asn Arg Phe Ser1 51097PRTMus musculus 109Asn Ala Lys Ser Leu Ala Glu1 51107PRTMus musculus 110Leu Val Ser Asn Leu Glu Ser1 51117PRTMus musculus 111Tyr Ala Thr Ser Leu Ala Asp1 51127PRTMus musculus 112Tyr Ala Ser Gln Ser Ile Ser1 51139PRTMus musculus 113Phe Gln Gly Ser His Val Pro Pro Thr1 51149PRTMus musculus 114Gln His His Tyr Gly Thr Pro Tyr Thr1 51159PRTMus musculus 115Gln His Ile Arg Glu Leu Thr Arg Ser1 51169PRTMus musculus 116Leu Gln His Gly Glu Ser Pro Phe Thr1 51179PRTMus musculus 117Gln Gln Ser Asp Thr Trp Pro Leu Thr1 51185PRTMus musculus 118Asp His Ala Leu His1 51195PRTMus musculus 119Asp His Ser Ile His1 51205PRTMus musculus 120Asn Tyr Val Ile His1 51219PRTMus musculus 121Ile Phe Pro Gly Asn Gly Asn Ile Glu1 51229PRTMus musculus 122Leu Phe Pro Gly Asn Gly Asn Phe Glu1 51239PRTMus musculus 123Ile Asn Pro Tyr Asn Asp Gly Ser Lys1 512411PRTMus musculus 124His Leu Ala Asn Thr Tyr Tyr Tyr Phe Asp Tyr1 5 101255PRTMus musculus 125Ser Asn Gly Asn Thr1 51265PRTMus musculus 126Glu Asn Ile Tyr Ser1 51275PRTMus musculus 127Thr Ser Gly Tyr Ser1 51285PRTMus musculus 128Gln Asp Ile Lys Ser1 51295PRTMus musculus 129Gln Asn Ile Gly Asn1 51304654DNAHomo sapiens 130ggcggcggct ggaggagagc gcggtggaga gccgagcggg cgggcggcgg gtgcggagcg 60ggcgagggag cgcgcgcggc cgccacaaag ctcgggcgcc gcggggctgc atgcggcgta 120cctggcccgg cgcggcgact gctctccggg ctggcggggg ccggccgcga gccccggggg 180ccccgaggcc gcagcttgcc tgcgcgctct gagccttcgc aactcgcgag caaagtttgg 240tggaggcaac gccaagcctg agtcctttct tcctctcgtt ccccaaatcc gagggcagcc 300cgcgggcgtc atgcccgcgc tcctccgcag cctggggtac gcgtgaagcc cgggaggctt 360ggcgccggcg aagacccaag gaccactctt ctgcgtttgg agttgctccc cgcaaccccg 420ggctcgtcgc tttctccatc ccgacccacg cggggcgcgg ggacaacaca ggtcgcggag 480gagcgttgcc attcaagtga ctgcagcagc agcggcagcg cctcggttcc tgagcccacc 540gcaggctgaa ggcattgcgc gtagtccatg cccgtagagg aagtgtgcag atgggattaa 600cgtccacatg gagatatgga agaggaccgg ggattggtac cgtaaccatg gtcagctggg 660gtcgtttcat ctgcctggtc gtggtcacca tggcaacctt gtccctggcc cggccctcct 720tcagtttagt tgaggatacc acattagagc cagaagagcc accaaccaaa taccaaatct 780ctcaaccaga agtgtacgtg gctgcgccag gggagtcgct agaggtgcgc tgcctgttga 840aagatgccgc cgtgatcagt tggactaagg atggggtgca cttggggccc aacaatagga 900cagtgcttat tggggagtac ttgcagataa agggcgccac gcctagagac tccggcctct 960atgcttgtac tgccagtagg actgtagaca gtgaaacttg gtacttcatg gtgaatgtca 1020cagatgccat ctcatccgga gatgatgagg atgacaccga tggtgcggaa gattttgtca 1080gtgagaacag taacaacaag agagcaccat actggaccaa cacagaaaag atggaaaagc 1140ggctccatgc tgtgcctgcg gccaacactg tcaagtttcg ctgcccagcc ggggggaacc 1200caatgccaac catgcggtgg ctgaaaaacg ggaaggagtt taagcaggag catcgcattg 1260gaggctacaa ggtacgaaac cagcactgga gcctcattat ggaaagtgtg gtcccatctg 1320acaagggaaa ttatacctgt gtagtggaga atgaatacgg gtccatcaat cacacgtacc 1380acctggatgt tgtggagcga tcgcctcacc ggcccatcct ccaagccgga ctgccggcaa 1440atgcctccac agtggtcgga ggagacgtag agtttgtctg caaggtttac agtgatgccc 1500agccccacat ccagtggatc aagcacgtgg aaaagaacgg cagtaaatac gggcccgacg 1560ggctgcccta cctcaaggtt ctcaaggccg ccggtgttaa caccacggac aaagagattg 1620aggttctcta tattcggaat gtaacttttg aggacgctgg ggaatatacg tgcttggcgg 1680gtaattctat tgggatatcc tttcactctg catggttgac agttctgcca gcgcctggaa 1740gagaaaagga gattacagct tccccagact acctggagat agccatttac tgcatagggg 1800tcttcttaat cgcctgtatg gtggtaacag tcatcctgtg ccgaatgaag aacacgacca 1860agaagccaga cttcagcagc cagccggctg tgcacaagct gaccaaacgt atccccctgc 1920ggagacaggt aacagtttcg gctgagtcca gctcctccat gaactccaac accccgctgg 1980tgaggataac aacacgcctc tcttcaacgg cagacacccc catgctggca ggggtctccg 2040agtatgaact tccagaggac ccaaaatggg agtttccaag agataagctg acactgggca 2100agcccctggg agaaggttgc tttgggcaag tggtcatggc ggaagcagtg ggaattgaca 2160aagacaagcc caaggaggcg gtcaccgtgg ccgtgaagat gttgaaagat gatgccacag 2220agaaagacct ttctgatctg gtgtcagaga tggagatgat gaagatgatt gggaaacaca 2280agaatatcat aaatcttctt ggagcctgca cacaggatgg gcctctctat gtcatagttg 2340agtatgcctc taaaggcaac ctccgagaat acctccgagc ccggaggcca cccgggatgg 2400agtactccta tgacattaac cgtgttcctg aggagcagat gaccttcaag gacttggtgt 2460catgcaccta ccagctggcc agaggcatgg agtacttggc ttcccaaaaa tgtattcatc 2520gagatttagc agccagaaat gttttggtaa cagaaaacaa tgtgatgaaa atagcagact 2580ttggactcgc cagagatatc aacaatatag actattacaa aaagaccacc aatgggcggc 2640ttccagtcaa gtggatggct ccagaagccc tgtttgatag agtatacact catcagagtg 2700atgtctggtc cttcggggtg ttaatgtggg agatcttcac tttagggggc tcgccctacc 2760cagggattcc cgtggaggaa ctttttaagc tgctgaagga aggacacaga atggataagc 2820cagccaactg caccaacgaa ctgtacatga tgatgaggga ctgttggcat gcagtgccct 2880cccagagacc aacgttcaag cagttggtag aagacttgga tcgaattctc actctcacaa 2940ccaatgagga atacttggac ctcagccaac ctctcgaaca gtattcacct agttaccctg 3000acacaagaag ttcttgttct tcaggagatg attctgtttt ttctccagac cccatgcctt 3060acgaaccatg ccttcctcag tatccacaca taaacggcag tgttaaaaca tgaatgactg 3120tgtctgcctg tccccaaaca ggacagcact gggaacctag ctacactgag cagggagacc 3180atgcctccca gagcttgttg tctccacttg tatatatgga tcagaggagt aaataattgg 3240aaaagtaatc agcatatgtg taaagattta tacagttgaa aacttgtaat cttccccagg 3300aggagaagaa ggtttctgga gcagtggact gccacaagcc accatgtaac ccctctcacc 3360tgccgtgcgt actggctgtg gaccagtagg actcaaggtg gacgtgcgtt ctgccttcct 3420tgttaatttt gtaataattg gagaagattt atgtcagcac acacttacag agcacaaatg 3480cagtatatag gtgctggatg tatgtaaata tattcaaatt atgtataaat atatattata 3540tatttacaag gagttatttt ttgtattgat tttaaatgga tgtcccaatg cacctagaaa 3600attggtctct ctttttttaa tagctatttg ctaaatgctg ttcttacaca taatttctta 3660attttcaccg agcagaggtg gaaaaatact tttgctttca gggaaaatgg tataacgtta 3720atttattaat aaattggtaa tatacaaaac aattaatcat ttatagtttt ttttgtaatt 3780taagtggcat ttctatgcag gcagcacagc agactagtta atctattgct tggacttaac 3840tagttatcag atcctttgaa aagagaatat ttacaatata tgactaattt ggggaaaatg 3900aagttttgat ttatttgtgt ttaaatgctg ctgtcagacg attgttctta gacctcctaa 3960atgccccata ttaaaagaac tcattcatag gaaggtgttt cattttggtg tgcaaccctg 4020tcattacgtc aacgcaacgt ctaactggac ttcccaagat aaatggtacc agcgtcctct 4080taaaagatgc cttaatccat tccttgagga cagaccttag ttgaaatgat agcagaatgt 4140gcttctctct ggcagctggc cttctgcttc tgagttgcac attaatcaga ttagcctgta 4200ttctcttcag tgaattttga taatggcttc cagactcttt ggcgttggag acgcctgtta 4260ggatcttcaa gtcccatcat agaaaattga aacacagagt tgttctgctg atagttttgg 4320ggatacgtcc atctttttaa gggattgctt tcatctaatt ctggcaggac ctcaccaaaa 4380gatccagcct catacctaca tcagacaaaa tatcgccgtt gttccttctg tactaaagta 4440ttgtgttttg ctttggaaac acccactcac tttgcaatag ccgtgcaaga tgaatgcaga 4500ttacactgat cttatgtgtt acaaaattgg agaaagtatt taataaaacc tgttaatttt 4560tatactgaca ataaaaatgt ttctacagat attaatgtta acaagacaaa ataaatgtca 4620cgcaacttat ttttttaata aaaaaaaaaa aaaa 46541314657DNAHomo sapiens 131ggcggcggct ggaggagagc gcggtggaga gccgagcggg cgggcggcgg gtgcggagcg 60ggcgagggag cgcgcgcggc cgccacaaag ctcgggcgcc gcggggctgc atgcggcgta 120cctggcccgg cgcggcgact gctctccggg ctggcggggg ccggccgcga gccccggggg 180ccccgaggcc gcagcttgcc tgcgcgctct gagccttcgc aactcgcgag caaagtttgg 240tggaggcaac gccaagcctg agtcctttct tcctctcgtt ccccaaatcc gagggcagcc 300cgcgggcgtc atgcccgcgc tcctccgcag cctggggtac gcgtgaagcc cgggaggctt 360ggcgccggcg aagacccaag gaccactctt ctgcgtttgg agttgctccc cgcaaccccg 420ggctcgtcgc tttctccatc ccgacccacg cggggcgcgg ggacaacaca ggtcgcggag 480gagcgttgcc attcaagtga ctgcagcagc agcggcagcg cctcggttcc tgagcccacc 540gcaggctgaa ggcattgcgc gtagtccatg cccgtagagg aagtgtgcag atgggattaa 600cgtccacatg gagatatgga agaggaccgg ggattggtac cgtaaccatg gtcagctggg 660gtcgtttcat ctgcctggtc gtggtcacca tggcaacctt gtccctggcc cggccctcct 720tcagtttagt tgaggatacc acattagagc cagaagagcc accaaccaaa taccaaatct 780ctcaaccaga agtgtacgtg gctgcgccag gggagtcgct agaggtgcgc tgcctgttga 840aagatgccgc cgtgatcagt tggactaagg atggggtgca cttggggccc aacaatagga 900cagtgcttat tggggagtac ttgcagataa agggcgccac gcctagagac tccggcctct 960atgcttgtac tgccagtagg actgtagaca gtgaaacttg gtacttcatg gtgaatgtca 1020cagatgccat ctcatccgga gatgatgagg atgacaccga tggtgcggaa gattttgtca 1080gtgagaacag taacaacaag agagcaccat actggaccaa cacagaaaag atggaaaagc 1140ggctccatgc tgtgcctgcg gccaacactg tcaagtttcg ctgcccagcc ggggggaacc 1200caatgccaac catgcggtgg ctgaaaaacg ggaaggagtt taagcaggag catcgcattg 1260gaggctacaa ggtacgaaac cagcactgga gcctcattat ggaaagtgtg gtcccatctg 1320acaagggaaa ttatacctgt gtagtggaga atgaatacgg gtccatcaat cacacgtacc 1380acctggatgt tgtggagcga tcgcctcacc ggcccatcct ccaagccgga ctgccggcaa 1440atgcctccac agtggtcgga ggagacgtag agtttgtctg caaggtttac agtgatgccc 1500agccccacat ccagtggatc aagcacgtgg aaaagaacgg cagtaaatac gggcccgacg 1560ggctgcccta cctcaaggtt ctcaagcact cggggataaa tagttccaat gcagaagtgc 1620tggctctgtt caatgtgacc gaggcggatg ctggggaata tatatgtaag gtctccaatt 1680atatagggca ggccaaccag tctgcctggc tcactgtcct gccaaaacag caagcgcctg 1740gaagagaaaa ggagattaca gcttccccag actacctgga gatagccatt tactgcatag 1800gggtcttctt aatcgcctgt atggtggtaa cagtcatcct gtgccgaatg aagaacacga 1860ccaagaagcc agacttcagc agccagccgg ctgtgcacaa gctgaccaaa cgtatccccc 1920tgcggagaca ggtaacagtt tcggctgagt ccagctcctc catgaactcc aacaccccgc 1980tggtgaggat aacaacacgc ctctcttcaa cggcagacac ccccatgctg gcaggggtct 2040ccgagtatga acttccagag gacccaaaat gggagtttcc aagagataag ctgacactgg 2100gcaagcccct gggagaaggt tgctttgggc aagtggtcat ggcggaagca gtgggaattg 2160acaaagacaa gcccaaggag gcggtcaccg tggccgtgaa gatgttgaaa gatgatgcca 2220cagagaaaga cctttctgat ctggtgtcag agatggagat gatgaagatg attgggaaac 2280acaagaatat cataaatctt cttggagcct gcacacagga tgggcctctc tatgtcatag 2340ttgagtatgc ctctaaaggc aacctccgag aatacctccg agcccggagg ccacccggga 2400tggagtactc ctatgacatt aaccgtgttc ctgaggagca gatgaccttc aaggacttgg 2460tgtcatgcac ctaccagctg

gccagaggca tggagtactt ggcttcccaa aaatgtattc 2520atcgagattt agcagccaga aatgttttgg taacagaaaa caatgtgatg aaaatagcag 2580actttggact cgccagagat atcaacaata tagactatta caaaaagacc accaatgggc 2640ggcttccagt caagtggatg gctccagaag ccctgtttga tagagtatac actcatcaga 2700gtgatgtctg gtccttcggg gtgttaatgt gggagatctt cactttaggg ggctcgccct 2760acccagggat tcccgtggag gaacttttta agctgctgaa ggaaggacac agaatggata 2820agccagccaa ctgcaccaac gaactgtaca tgatgatgag ggactgttgg catgcagtgc 2880cctcccagag accaacgttc aagcagttgg tagaagactt ggatcgaatt ctcactctca 2940caaccaatga ggaatacttg gacctcagcc aacctctcga acagtattca cctagttacc 3000ctgacacaag aagttcttgt tcttcaggag atgattctgt tttttctcca gaccccatgc 3060cttacgaacc atgccttcct cagtatccac acataaacgg cagtgttaaa acatgaatga 3120ctgtgtctgc ctgtccccaa acaggacagc actgggaacc tagctacact gagcagggag 3180accatgcctc ccagagcttg ttgtctccac ttgtatatat ggatcagagg agtaaataat 3240tggaaaagta atcagcatat gtgtaaagat ttatacagtt gaaaacttgt aatcttcccc 3300aggaggagaa gaaggtttct ggagcagtgg actgccacaa gccaccatgt aacccctctc 3360acctgccgtg cgtactggct gtggaccagt aggactcaag gtggacgtgc gttctgcctt 3420ccttgttaat tttgtaataa ttggagaaga tttatgtcag cacacactta cagagcacaa 3480atgcagtata taggtgctgg atgtatgtaa atatattcaa attatgtata aatatatatt 3540atatatttac aaggagttat tttttgtatt gattttaaat ggatgtccca atgcacctag 3600aaaattggtc tctctttttt taatagctat ttgctaaatg ctgttcttac acataatttc 3660ttaattttca ccgagcagag gtggaaaaat acttttgctt tcagggaaaa tggtataacg 3720ttaatttatt aataaattgg taatatacaa aacaattaat catttatagt tttttttgta 3780atttaagtgg catttctatg caggcagcac agcagactag ttaatctatt gcttggactt 3840aactagttat cagatccttt gaaaagagaa tatttacaat atatgactaa tttggggaaa 3900atgaagtttt gatttatttg tgtttaaatg ctgctgtcag acgattgttc ttagacctcc 3960taaatgcccc atattaaaag aactcattca taggaaggtg tttcattttg gtgtgcaacc 4020ctgtcattac gtcaacgcaa cgtctaactg gacttcccaa gataaatggt accagcgtcc 4080tcttaaaaga tgccttaatc cattccttga ggacagacct tagttgaaat gatagcagaa 4140tgtgcttctc tctggcagct ggccttctgc ttctgagttg cacattaatc agattagcct 4200gtattctctt cagtgaattt tgataatggc ttccagactc tttggcgttg gagacgcctg 4260ttaggatctt caagtcccat catagaaaat tgaaacacag agttgttctg ctgatagttt 4320tggggatacg tccatctttt taagggattg ctttcatcta attctggcag gacctcacca 4380aaagatccag cctcatacct acatcagaca aaatatcgcc gttgttcctt ctgtactaaa 4440gtattgtgtt ttgctttgga aacacccact cactttgcaa tagccgtgca agatgaatgc 4500agattacact gatcttatgt gttacaaaat tggagaaagt atttaataaa acctgttaat 4560ttttatactg acaataaaaa tgtttctaca gatattaatg ttaacaagac aaaataaatg 4620tcacgcaact tattttttta ataaaaaaaa aaaaaaa 46571322781DNAHomo sapiens 132tgactgcagc agcagcggca gcgcctcggt tcctgagccc accgcaggct gaaggcattg 60cgcgtagtcc atgcccgtag aggaagtgtg cagatgggat taacgtccac atggagatat 120ggaagaggac cggggattgg taccgtaacc atggtcagct ggggtcgttt catctgcctg 180gtcgtggtca ccatggcaac cttgtccctg gcccggccct ccttcagttt agttgaggat 240accacattag agccagaaga gccaccaacc aaataccaaa tctctcaacc agaagtgtac 300gtggctgcgc caggggagtc gctagaggtg cgctgcctgt tgaaagatgc cgccgtgatc 360agttggacta aggatggggt gcacttgggg cccaacaata ggacagtgct tattggggag 420tacttgcaga taaagggcgc cacgcctaga gactccggcc tctatgcttg tactgccagt 480aggactgtag acagtgaaac ttggtacttc atggtgaatg tcacagatgc catctcatcc 540ggagatgatg aggatgacac cgatggtgcg gaagattttg tcagtgagaa cagtaacaac 600aagagagcac catactggac caacacagaa aagatggaaa agcggctcca tgctgtgcct 660gcggccaaca ctgtcaagtt tcgctgccca gccgggggga acccaatgcc aaccatgcgg 720tggctgaaaa acgggaagga gtttaagcag gagcatcgca ttggaggcta caaggtacga 780aaccagcact ggagcctcat tatggaaagt gtggtcccat ctgacaaggg aaattatacc 840tgtgtagtgg agaatgaata cgggtccatc aatcacacgt accacctgga tgttgtggag 900cgatcgcctc accggcccat cctccaagcc ggactgccgg caaatgcctc cacagtggtc 960ggaggagacg tagagtttgt ctgcaaggtt tacagtgatg cccagcccca catccagtgg 1020atcaagcacg tggaaaagaa cggcagtaaa tacgggcccg acgggctgcc ctacctcaag 1080gttctcaagc actcggggat aaatagttcc aatgcagaag tgctggctct gttcaatgtg 1140accgaggcgg atgctgggga atatatatgt aaggtctcca attatatagg gcaggccaac 1200cagtctgcct ggctcactgt cctgccaaaa cagcaagcgc ctggaagaga aaaggagatt 1260acagcttccc cagactacct ggagatagcc atttactgca taggggtctt cttaatcgcc 1320tgtatggtgg taacagtcat cctgtgccga atgaagaaca cgaccaagaa gccagacttc 1380agcagccagc cggctgtgca caagctgacc aaacgtatcc ccctgcggag acaggtaaca 1440gtttcggctg agtccagctc ctccatgaac tccaacaccc cgctggtgag gataacaaca 1500cgcctctctt caacggcaga cacccccatg ctggcagggg tctccgagta tgaacttcca 1560gaggacccaa aatgggagtt tccaagagat aagctgacac tgggcaagcc cctgggagaa 1620ggttgctttg ggcaagtggt catggcggaa gcagtgggaa ttgacaaaga caagcccaag 1680gaggcggtca ccgtggccgt gaagatgttg aaagatgatg ccacagagaa agacctttct 1740gatctggtgt cagagatgga gatgatgaag atgattggga aacacaagaa tatcataaat 1800cttcttggag cctgcacaca ggatgggcct ctctatgtca tagttgagta tgcctctaaa 1860ggcaacctcc gagaatacct ccgagcccgg aggccacccg ggatggagta ctcctatgac 1920attaaccgtg ttcctgagga gcagatgacc ttcaaggact tggtgtcatg cacctaccag 1980ctggccagag gcatggagta cttggcttcc caaaaatgta ttcatcgaga tttagcagcc 2040agaaatgttt tggtaacaga aaacaatgtg atgaaaatag cagactttgg actcgccaga 2100gatatcaaca atatagacta ttacaaaaag accaccaatg ggcggcttcc agtcaagtgg 2160atggctccag aagccctgtt tgatagagta tacactcatc agagtgatgt ctggtccttc 2220ggggtgttaa tgtgggagat cttcacttta gggggctcgc cctacccagg gattcccgtg 2280gaggaacttt ttaagctgct gaaggaagga cacagaatgg ataagccagc caactgcacc 2340aacgaactgt acatgatgat gagggactgt tggcatgcag tgccctccca gagaccaacg 2400ttcaagcagt tggtagaaga cttggatcga attctcactc tcacaaccaa tgagatctga 2460aagtttatgg cttcattgag aaactgggaa aagttggtca ggcgcagtgg ctcatgcctg 2520taatcccagc actttgggag gccgaggcag gcggatcatg aggtcaggag ttccagacca 2580gcctggccaa catggtgaaa ccctgtctct actaaagata caaaaaatta gccgggcgtg 2640ttggtgtgca cctgtaatcc cagctactcc gggaggctga ggcaggagag tcacttgaac 2700cggggaggcg gaggttgcag tgagccgaga tcatgccatt gcattccagc cttggcgaca 2760gagcgagact ccgtctcaaa a 27811333821DNAHomo sapiens 133tgactgcagc agcagcggca gcgcctcggt tcctgagccc accgcaggct gaaggcattg 60cgcgtagtcc atgcccgtag aggaagtgtg cagatgggat taacgtccac atggagatat 120ggaagaggac cggggattgg taccgtaacc atggtcagct ggggtcgttt catctgcctg 180gtcgtggtca ccatggcaac cttgtccctg gcccggccct ccttcagttt agttgaggat 240accacattag agccagaaga gccaccaacc aaataccaaa tctctcaacc agaagtgtac 300gtggctgcgc caggggagtc gctagaggtg cgctgcctgt tgaaagatgc cgccgtgatc 360agttggacta aggatggggt gcacttgggg cccaacaata ggacagtgct tattggggag 420tacttgcaga taaagggcgc cacgcctaga gactccggcc tctatgcttg tactgccagt 480aggactgtag acagtgaaac ttggtacttc atggtgaatg tcacagatgc catctcatcc 540ggagatgatg aggatgacac cgatggtgcg gaagattttg tcagtgagaa cagtaacaac 600aagagagcac catactggac caacacagaa aagatggaaa agcggctcca tgctgtgcct 660gcggccaaca ctgtcaagtt tcgctgccca gccgggggga acccaatgcc aaccatgcgg 720tggctgaaaa acgggaagga gtttaagcag gagcatcgca ttggaggcta caaggtacga 780aaccagcact ggagcctcat tatggaaagt gtggtcccat ctgacaaggg aaattatacc 840tgtgtagtgg agaatgaata cgggtccatc aatcacacgt accacctgga tgttgtggcg 900cctggaagag aaaaggagat tacagcttcc ccagactacc tggagatagc catttactgc 960ataggggtct tcttaatcgc ctgtatggtg gtaacagtca tcctgtgccg aatgaagaac 1020acgaccaaga agccagactt cagcagccag ccggctgtgc acaagctgac caaacgtatc 1080cccctgcgga gacaggtaac agtttcggct gagtccagct cctccatgaa ctccaacacc 1140ccgctggtga ggataacaac acgcctctct tcaacggcag acacccccat gctggcaggg 1200gtctccgagt atgaacttcc agaggaccca aaatgggagt ttccaagaga taagctgaca 1260ctgggcaagc ccctgggaga aggttgcttt gggcaagtgg tcatggcgga agcagtggga 1320attgacaaag acaagcccaa ggaggcggtc accgtggccg tgaagatgtt gaaagatgat 1380gccacagaga aagacctttc tgatctggtg tcagagatgg agatgatgaa gatgattggg 1440aaacacaaga atatcataaa tcttcttgga gcctgcacac aggatgggcc tctctatgtc 1500atagttgagt atgcctctaa aggcaacctc cgagaatacc tccgagcccg gaggccaccc 1560gggatggagt actcctatga cattaaccgt gttcctgagg agcagatgac cttcaaggac 1620ttggtgtcat gcacctacca gctggccaga ggcatggagt acttggcttc ccaaaaatgt 1680attcatcgag atttagcagc cagaaatgtt ttggtaacag aaaacaatgt gatgaaaata 1740gcagactttg gactcgccag agatatcaac aatatagact attacaaaaa gaccaccaat 1800gggcggcttc cagtcaagtg gatggctcca gaagccctgt ttgatagagt atacactcat 1860cagagtgatg tctggtcctt cggggtgtta atgtgggaga tcttcacttt agggggctcg 1920ccctacccag ggattcccgt ggaggaactt tttaagctgc tgaaggaagg acacagaatg 1980gataagccag ccaactgcac caacgaactg tacatgatga tgagggactg ttggcatgca 2040gtgccctccc agagaccaac gttcaagcag ttggtagaag acttggatcg aattctcact 2100ctcacaacca atgaggaata cttggacctc agccaacctc tcgaacagta ttcacctagt 2160taccctgaca caagaagttc ttgttcttca ggagatgatt ctgttttttc tccagacccc 2220atgccttacg aaccatgcct tcctcagtat ccacacataa acggcagtgt taaaacatga 2280atgactgtgt ctgcctgtcc ccaaacagga cagcactggg aacctagcta cactgagcag 2340ggagaccatg cctcccagag cttgttgtct ccacttgtat atatggatca gaggagtaaa 2400taattggaaa agtaatcagc atatgtgtaa agatttatac agttgaaaac ttgtaatctt 2460ccccaggagg agaagaaggt ttctggagca gtggactgcc acaagccacc atgtaacccc 2520tctcacctgc cgtgcgtact ggctgtggac cagtaggact caaggtggac gtgcgttctg 2580ccttccttgt taattttgta ataattggag aagatttatg tcagcacaca cttacagagc 2640acaaatgcag tatataggtg ctggatgtat gtaaatatat tcaaattatg tataaatata 2700tattatatat ttacaaggag ttattttttg tattgatttt aaatggatgt cccaatgcac 2760ctagaaaatt ggtctctctt tttttaatag ctatttgcta aatgctgttc ttacacataa 2820tttcttaatt ttcaccgagc agaggtggaa aaatactttt gctttcaggg aaaatggtat 2880aacgttaatt tattaataaa ttggtaatat acaaaacaat taatcattta tagttttttt 2940tgtaatttaa gtggcatttc tatgcaggca gcacagcaga ctagttaatc tattgcttgg 3000acttaactag ttatcagatc ctttgaaaag agaatattta caatatatga ctaatttggg 3060gaaaatgaag ttttgattta tttgtgttta aatgctgctg tcagacgatt gttcttagac 3120ctcctaaatg ccccatatta aaagaactca ttcataggaa ggtgtttcat tttggtgtgc 3180aaccctgtca ttacgtcaac gcaacgtcta actggacttc ccaagataaa tggtaccagc 3240gtcctcttaa aagatgcctt aatccattcc ttgaggacag accttagttg aaatgatagc 3300agaatgtgct tctctctggc agctggcctt ctgcttctga gttgcacatt aatcagatta 3360gcctgtattc tcttcagtga attttgataa tggcttccag actctttggc gttggagacg 3420cctgttagga tcttcaagtc ccatcataga aaattgaaac acagagttgt tctgctgata 3480gttttgggga tacgtccatc tttttaaggg attgctttca tctaattctg gcaggacctc 3540accaaaagat ccagcctcat acctacatca gacaaaatat cgccgttgtt ccttctgtac 3600taaagtattg tgttttgctt tggaaacacc cactcacttt gcaatagccg tgcaagatga 3660atgcagatta cactgatctt atgtgttaca aaattggaga aagtatttaa taaaacctgt 3720taatttttat actgacaata aaaatgtttc tacagatatt aatgttaaca agacaaaata 3780aatgtcacgc aacttatttt tttaataaaa aaaaaaaaaa a 38211343708DNAHomo sapiens 134aatttgttga ggaatttccc cctagccttg accccttgac agctcccgct cctactcagt 60gctggggaga agtagggagg ccttaagcga agagatgggt ctgcactttg gaggagccgg 120acactgttga ctttcctgat gtgaaatcta cccaggaaca aaacaccagt gactgcagca 180gcagcggcag cgcctcggtt cctgagccca ccgcaggctg aaggcattgc gcgtagtcca 240tgcccgtaga ggaagtgtgc agatgggatt aacgtccaca tggagatatg gaagaggacc 300ggggattggt accgtaacca tggtcagctg gggtcgtttc atctgcctgg tcgtggtcac 360catggcaacc ttgtccctgg cccggccctc cttcagttta gttgaggata ccacattaga 420gccagaagat gccatctcat ccggagatga tgaggatgac accgatggtg cggaagattt 480tgtcagtgag aacagtaaca acaagagagc accatactgg accaacacag aaaagatgga 540aaagcggctc catgctgtgc ctgcggccaa cactgtcaag tttcgctgcc cagccggggg 600gaacccaatg ccaaccatgc ggtggctgaa aaacgggaag gagtttaagc aggagcatcg 660cattggaggc tacaaggtac gaaaccagca ctggagcctc attatggaaa gtgtggtccc 720atctgacaag ggaaattata cctgtgtagt ggagaatgaa tacgggtcca tcaatcacac 780gtaccacctg gatgttgtgg agcgatcgcc tcaccggccc atcctccaag ccggactgcc 840ggcaaatgcc tccacagtgg tcggaggaga cgtagagttt gtctgcaagg tttacagtga 900tgcccagccc cacatccagt ggatcaagca cgtggaaaag aacggcagta aatacgggcc 960cgacgggctg ccctacctca aggttctcaa ggccgccggt gttaacacca cggacaaaga 1020gattgaggtt ctctatattc ggaatgtaac ttttgaggac gctggggaat atacgtgctt 1080ggcgggtaat tctattggga tatcctttca ctctgcatgg ttgacagttc tgccagcgcc 1140tggaagagaa aaggagatta cagcttcccc agactacctg gagatagcca tttactgcat 1200aggggtcttc ttaatcgcct gtatggtggt aacagtcatc ctgtgccgaa tgaagaacac 1260gaccaagaag ccagacttca gcagccagcc ggctgtgcac aagctgacca aacgtatccc 1320cctgcggaga caggtaacag tttcggctga gtccagctcc tccatgaact ccaacacccc 1380gctggtgagg ataacaacac gcctctcttc aacggcagac acccccatgc tggcaggggt 1440ctccgagtat gaacttccag aggacccaaa atgggagttt ccaagagata agctgacact 1500gggcaagccc ctgggagaag gttgctttgg gcaagtggtc atggcggaag cagtgggaat 1560tgacaaagac aagcccaagg aggcggtcac cgtggccgtg aagatgttga aagatgatgc 1620cacagagaaa gacctttctg atctggtgtc agagatggag atgatgaaga tgattgggaa 1680acacaagaat atcataaatc ttcttggagc ctgcacacag gatgggcctc tctatgtcat 1740agttgagtat gcctctaaag gcaacctccg agaatacctc cgagcccgga ggccacccgg 1800gatggagtac tcctatgaca ttaaccgtgt tcctgaggag cagatgacct tcaaggactt 1860ggtgtcatgc acctaccagc tggccagagg catggagtac ttggcttccc aaaaatgtat 1920tcatcgagat ttagcagcca gaaatgtttt ggtaacagaa aacaatgtga tgaaaatagc 1980agactttgga ctcgccagag atatcaacaa tatagactat tacaaaaaga ccaccaatgg 2040gcggcttcca gtcaagtgga tggctccaga agccctgttt gatagagtat acactcatca 2100gagtgatgtc tggtccttcg gggtgttaat gtgggagatc ttcactttag ggggctcgcc 2160ctacccaggg attcccgtgg aggaactttt taagctgctg aaggaaggac acagaatgga 2220taagccagcc aactgcacca acgaactgta catgatgatg agggactgtt ggcatgcagt 2280gccctcccag agaccaacgt tcaagcagtt ggtagaagac ttggatcgaa ttctcactct 2340cacaaccaat gaggaggaga agaaggtttc tggagcagtg gactgccaca agccaccatg 2400taacccctct cacctgccgt gcgtactggc tgtggaccag taggactcaa ggtggacgtg 2460cgttctgcct tccttgttaa ttttgtaata attggagaag atttatgtca gcacacactt 2520acagagcaca aatgcagtat ataggtgctg gatgtatgta aatatattca aattatgtat 2580aaatatatat tatatattta caaggagtta ttttttgtat tgattttaaa tggatgtccc 2640aatgcaccta gaaaattggt ctctcttttt ttaatagcta tttgctaaat gctgttctta 2700cacataattt cttaattttc accgagcaga ggtggaaaaa tacttttgct ttcagggaaa 2760atggtataac gttaatttat taataaattg gtaatataca aaacaattaa tcatttatag 2820ttttttttgt aatttaagtg gcatttctat gcaggcagca cagcagacta gttaatctat 2880tgcttggact taactagtta tcagatcctt tgaaaagaga atatttacaa tatatgacta 2940atttggggaa aatgaagttt tgatttattt gtgtttaaat gctgctgtca gacgattgtt 3000cttagacctc ctaaatgccc catattaaaa gaactcattc ataggaaggt gtttcatttt 3060ggtgtgcaac cctgtcatta cgtcaacgca acgtctaact ggacttccca agataaatgg 3120taccagcgtc ctcttaaaag atgccttaat ccattccttg aggacagacc ttagttgaaa 3180tgatagcaga atgtgcttct ctctggcagc tggccttctg cttctgagtt gcacattaat 3240cagattagcc tgtattctct tcagtgaatt ttgataatgg cttccagact ctttggcgtt 3300ggagacgcct gttaggatct tcaagtccca tcatagaaaa ttgaaacaca gagttgttct 3360gctgatagtt ttggggatac gtccatcttt ttaagggatt gctttcatct aattctggca 3420ggacctcacc aaaagatcca gcctcatacc tacatcagac aaaatatcgc cgttgttcct 3480tctgtactaa agtattgtgt tttgctttgg aaacacccac tcactttgca atagccgtgc 3540aagatgaatg cagattacac tgatcttatg tgttacaaaa ttggagaaag tatttaataa 3600aacctgttaa tttttatact gacaataaaa atgtttctac agatattaat gttaacaaga 3660caaaataaat gtcacgcaac ttattttttt aataaaaaaa aaaaaaaa 37081354103DNAHomo sapiens 135gagcacacat tgcctcactg aagtggctgc acgtatctga gtcctgtagc tactgtttta 60tctctgtttc ttaaaagtat gcttttaaaa agattagcct cacacatttc tgtggaccgg 120tctggtggta tcacctggga ctctgaggtg aggatggaag gatttagcag ataatgaaaa 180agaactctgt ttgcgcacat ttgagaggct gaaaaatggt tttatcccac ttgggctgga 240gtgatttggc attggggaag attccctgac tcgccaatct ctttccttta gtgactgcag 300cagcagcggc agcgcctcgg ttcctgagcc caccgcaggc tgaaggcatt gcgcgtagtc 360catgcccgta gaggaagtgt gcagatggga ttaacgtcca catggagata tggaagagga 420ccggggattg gtaccgtaac catggtcagc tggggtcgtt tcatctgcct ggtcgtggtc 480accatggcaa ccttgtccct ggcccggccc tccttcagtt tagttgagga taccacatta 540gagccagaag gagcaccata ctggaccaac acagaaaaga tggaaaagcg gctccatgct 600gtgcctgcgg ccaacactgt caagtttcgc tgcccagccg gggggaaccc aatgccaacc 660atgcggtggc tgaaaaacgg gaaggagttt aagcaggagc atcgcattgg aggctacaag 720gtacgaaacc agcactggag cctcattatg gaaagtgtgg tcccatctga caagggaaat 780tatacctgtg tagtggagaa tgaatacggg tccatcaatc acacgtacca cctggatgtt 840gtggagcgat cgcctcaccg gcccatcctc caagccggac tgccggcaaa tgcctccaca 900gtggtcggag gagacgtaga gtttgtctgc aaggtttaca gtgatgccca gccccacatc 960cagtggatca agcacgtgga aaagaacggc agtaaatacg ggcccgacgg gctgccctac 1020ctcaaggttc tcaaggccgc cggtgttaac accacggaca aagagattga ggttctctat 1080attcggaatg taacttttga ggacgctggg gaatatacgt gcttggcggg taattctatt 1140gggatatcct ttcactctgc atggttgaca gttctgccag cgcctggaag agaaaaggag 1200attacagctt ccccagacta cctggagata gccatttact gcataggggt cttcttaatc 1260gcctgtatgg tggtaacagt catcctgtgc cgaatgaaga acacgaccaa gaagccagac 1320ttcagcagcc agccggctgt gcacaagctg accaaacgta tccccctgcg gagacaggta 1380acagtttcgg ctgagtccag ctcctccatg aactccaaca ccccgctggt gaggataaca 1440acacgcctct cttcaacggc agacaccccc atgctggcag gggtctccga gtatgaactt 1500ccagaggacc caaaatggga gtttccaaga gataagctga cactgggcaa gcccctggga 1560gaaggttgct ttgggcaagt ggtcatggcg gaagcagtgg gaattgacaa agacaagccc 1620aaggaggcgg tcaccgtggc cgtgaagatg ttgaaagatg atgccacaga gaaagacctt 1680tctgatctgg tgtcagagat ggagatgatg aagatgattg ggaaacacaa gaatatcata 1740aatcttcttg gagcctgcac acaggatggg cctctctatg tcatagttga gtatgcctct 1800aaaggcaacc tccgagaata cctccgagcc cggaggccac ccgggatgga gtactcctat 1860gacattaacc gtgttcctga ggagcagatg accttcaagg acttggtgtc atgcacctac 1920cagctggcca gaggcatgga gtacttggct tcccaaaaat gtattcatcg agatttagca 1980gccagaaatg ttttggtaac agaaaacaat gtgatgaaaa tagcagactt tggactcgcc 2040agagatatca acaatataga ctattacaaa aagaccacca atgggcggct tccagtcaag 2100tggatggctc cagaagccct gtttgataga gtatacactc atcagagtga tgtctggtcc 2160ttcggggtgt taatgtggga gatcttcact ttagggggct cgccctaccc agggattccc 2220gtggaggaac tttttaagct gctgaaggaa ggacacagaa tggataagcc agccaactgc 2280accaacgaac tgtacatgat gatgagggac tgttggcatg cagtgccctc ccagagacca 2340acgttcaagc agttggtaga agacttggat cgaattctca ctctcacaac caatgaggaa

2400tacttggacc tcagccaacc tctcgaacag tattcaccta gttaccctga cacaagaagt 2460tcttgttctt caggagatga ttctgttttt tctccagacc ccatgcctta cgaaccatgc 2520cttcctcagt atccacacat aaacggcagt gttaaaacat gaatgactgt gtctgcctgt 2580ccccaaacag gacagcactg ggaacctagc tacactgagc agggagacca tgcctcccag 2640agcttgttgt ctccacttgt atatatggat cagaggagta aataattgga aaagtaatca 2700gcatatgtgt aaagatttat acagttgaaa acttgtaatc ttccccagga ggagaagaag 2760gtttctggag cagtggactg ccacaagcca ccatgtaacc cctctcacct gccgtgcgta 2820ctggctgtgg accagtagga ctcaaggtgg acgtgcgttc tgccttcctt gttaattttg 2880taataattgg agaagattta tgtcagcaca cacttacaga gcacaaatgc agtatatagg 2940tgctggatgt atgtaaatat attcaaatta tgtataaata tatattatat atttacaagg 3000agttattttt tgtattgatt ttaaatggat gtcccaatgc acctagaaaa ttggtctctc 3060tttttttaat agctatttgc taaatgctgt tcttacacat aatttcttaa ttttcaccga 3120gcagaggtgg aaaaatactt ttgctttcag ggaaaatggt ataacgttaa tttattaata 3180aattggtaat atacaaaaca attaatcatt tatagttttt tttgtaattt aagtggcatt 3240tctatgcagg cagcacagca gactagttaa tctattgctt ggacttaact agttatcaga 3300tcctttgaaa agagaatatt tacaatatat gactaatttg gggaaaatga agttttgatt 3360tatttgtgtt taaatgctgc tgtcagacga ttgttcttag acctcctaaa tgccccatat 3420taaaagaact cattcatagg aaggtgtttc attttggtgt gcaaccctgt cattacgtca 3480acgcaacgtc taactggact tcccaagata aatggtacca gcgtcctctt aaaagatgcc 3540ttaatccatt ccttgaggac agaccttagt tgaaatgata gcagaatgtg cttctctctg 3600gcagctggcc ttctgcttct gagttgcaca ttaatcagat tagcctgtat tctcttcagt 3660gaattttgat aatggcttcc agactctttg gcgttggaga cgcctgttag gatcttcaag 3720tcccatcata gaaaattgaa acacagagtt gttctgctga tagttttggg gatacgtcca 3780tctttttaag ggattgcttt catctaattc tggcaggacc tcaccaaaag atccagcctc 3840atacctacat cagacaaaat atcgccgttg ttccttctgt actaaagtat tgtgttttgc 3900tttggaaaca cccactcact ttgcaatagc cgtgcaagat gaatgcagat tacactgatc 3960ttatgtgtta caaaattgga gaaagtattt aataaaacct gttaattttt atactgacaa 4020taaaaatgtt tctacagata ttaatgttaa caagacaaaa taaatgtcac gcaacttatt 4080tttttaataa aaaaaaaaaa aaa 41031364306DNAHomo sapiens 136ggcggcggct ggaggagagc gcggtggaga gccgagcggg cgggcggcgg gtgcggagcg 60ggcgagggag cgcgcgcggc cgccacaaag ctcgggcgcc gcggggctgc atgcggcgta 120cctggcccgg cgcggcgact gctctccggg ctggcggggg ccggccgcga gccccggggg 180ccccgaggcc gcagcttgcc tgcgcgctct gagccttcgc aactcgcgag caaagtttgg 240tggaggcaac gccaagcctg agtcctttct tcctctcgtt ccccaaatcc gagggcagcc 300cgcgggcgtc atgcccgcgc tcctccgcag cctggggtac gcgtgaagcc cgggaggctt 360ggcgccggcg aagacccaag gaccactctt ctgcgtttgg agttgctccc cgcaaccccg 420ggctcgtcgc tttctccatc ccgacccacg cggggcgcgg ggacaacaca ggtcgcggag 480gagcgttgcc attcaagtga ctgcagcagc agcggcagcg cctcggttcc tgagcccacc 540gcaggctgaa ggcattgcgc gtagtccatg cccgtagagg aagtgtgcag atgggattaa 600cgtccacatg gagatatgga agaggaccgg ggattggtac cgtaaccatg gtcagctggg 660gtcgtttcat ctgcctggtc gtggtcacca tggcaacctt gtccctggcc cggccctcct 720tcagtttagt tgaggatacc acattagagc cagaagagcc accaaccaaa taccaaatct 780ctcaaccaga agtgtacgtg gctgcgccag gggagtcgct agaggtgcgc tgcctgttga 840aagatgccgc cgtgatcagt tggactaagg atggggtgca cttggggccc aacaatagga 900cagtgcttat tggggagtac ttgcagataa agggcgccac gcctagagac tccggcctct 960atgcttgtac tgccagtagg actgtagaca gtgaaacttg gtacttcatg gtgaatgtca 1020cagatgccat ctcatccgga gatgatgagg atgacaccga tggtgcggaa gattttgtca 1080gtgagaacag taacaacaag agagcaccat actggaccaa cacagaaaag atggaaaagc 1140ggctccatgc tgtgcctgcg gccaacactg tcaagtttcg ctgcccagcc ggggggaacc 1200caatgccaac catgcggtgg ctgaaaaacg ggaaggagtt taagcaggag catcgcattg 1260gaggctacaa ggtacgaaac cagcactgga gcctcattat ggaaagtgtg gtcccatctg 1320acaagggaaa ttatacctgt gtagtggaga atgaatacgg gtccatcaat cacacgtacc 1380acctggatgt tgtggagcga tcgcctcacc ggcccatcct ccaagccgga ctgccggcaa 1440atgcctccac agtggtcgga ggagacgtag agtttgtctg caaggtttac agtgatgccc 1500agccccacat ccagtggatc aagcacgtgg aaaagaacgg cagtaaatac gggcccgacg 1560ggctgcccta cctcaaggtt ctcaaggttt cggctgagtc cagctcctcc atgaactcca 1620acaccccgct ggtgaggata acaacacgcc tctcttcaac ggcagacacc cccatgctgg 1680caggggtctc cgagtatgaa cttccagagg acccaaaatg ggagtttcca agagataagc 1740tgacactggg caagcccctg ggagaaggtt gctttgggca agtggtcatg gcggaagcag 1800tgggaattga caaagacaag cccaaggagg cggtcaccgt ggccgtgaag atgttgaaag 1860atgatgccac agagaaagac ctttctgatc tggtgtcaga gatggagatg atgaagatga 1920ttgggaaaca caagaatatc ataaatcttc ttggagcctg cacacaggat gggcctctct 1980atgtcatagt tgagtatgcc tctaaaggca acctccgaga atacctccga gcccggaggc 2040cacccgggat ggagtactcc tatgacatta accgtgttcc tgaggagcag atgaccttca 2100aggacttggt gtcatgcacc taccagctgg ccagaggcat ggagtacttg gcttcccaaa 2160aatgtattca tcgagattta gcagccagaa atgttttggt aacagaaaac aatgtgatga 2220aaatagcaga ctttggactc gccagagata tcaacaatat agactattac aaaaagacca 2280ccaatgggcg gcttccagtc aagtggatgg ctccagaagc cctgtttgat agagtataca 2340ctcatcagag tgatgtctgg tccttcgggg tgttaatgtg ggagatcttc actttagggg 2400gctcgcccta cccagggatt cccgtggagg aactttttaa gctgctgaag gaaggacaca 2460gaatggataa gccagccaac tgcaccaacg aactgtacat gatgatgagg gactgttggc 2520atgcagtgcc ctcccagaga ccaacgttca agcagttggt agaagacttg gatcgaattc 2580tcactctcac aaccaatgag gaatacttgg acctcagcca acctctcgaa cagtattcac 2640ctagttaccc tgacacaaga agttcttgtt cttcaggaga tgattctgtt ttttctccag 2700accccatgcc ttacgaacca tgccttcctc agtatccaca cataaacggc agtgttaaaa 2760catgaatgac tgtgtctgcc tgtccccaaa caggacagca ctgggaacct agctacactg 2820agcagggaga ccatgcctcc cagagcttgt tgtctccact tgtatatatg gatcagagga 2880gtaaataatt ggaaaagtaa tcagcatatg tgtaaagatt tatacagttg aaaacttgta 2940atcttcccca ggaggagaag aaggtttctg gagcagtgga ctgccacaag ccaccatgta 3000acccctctca cctgccgtgc gtactggctg tggaccagta ggactcaagg tggacgtgcg 3060ttctgccttc cttgttaatt ttgtaataat tggagaagat ttatgtcagc acacacttac 3120agagcacaaa tgcagtatat aggtgctgga tgtatgtaaa tatattcaaa ttatgtataa 3180atatatatta tatatttaca aggagttatt ttttgtattg attttaaatg gatgtcccaa 3240tgcacctaga aaattggtct ctcttttttt aatagctatt tgctaaatgc tgttcttaca 3300cataatttct taattttcac cgagcagagg tggaaaaata cttttgcttt cagggaaaat 3360ggtataacgt taatttatta ataaattggt aatatacaaa acaattaatc atttatagtt 3420ttttttgtaa tttaagtggc atttctatgc aggcagcaca gcagactagt taatctattg 3480cttggactta actagttatc agatcctttg aaaagagaat atttacaata tatgactaat 3540ttggggaaaa tgaagttttg atttatttgt gtttaaatgc tgctgtcaga cgattgttct 3600tagacctcct aaatgcccca tattaaaaga actcattcat aggaaggtgt ttcattttgg 3660tgtgcaaccc tgtcattacg tcaacgcaac gtctaactgg acttcccaag ataaatggta 3720ccagcgtcct cttaaaagat gccttaatcc attccttgag gacagacctt agttgaaatg 3780atagcagaat gtgcttctct ctggcagctg gccttctgct tctgagttgc acattaatca 3840gattagcctg tattctcttc agtgaatttt gataatggct tccagactct ttggcgttgg 3900agacgcctgt taggatcttc aagtcccatc atagaaaatt gaaacacaga gttgttctgc 3960tgatagtttt ggggatacgt ccatcttttt aagggattgc tttcatctaa ttctggcagg 4020acctcaccaa aagatccagc ctcataccta catcagacaa aatatcgccg ttgttccttc 4080tgtactaaag tattgtgttt tgctttggaa acacccactc actttgcaat agccgtgcaa 4140gatgaatgca gattacactg atcttatgtg ttacaaaatt ggagaaagta tttaataaaa 4200cctgttaatt tttatactga caataaaaat gtttctacag atattaatgt taacaagaca 4260aaataaatgt cacgcaactt atttttttaa taaaaaaaaa aaaaaa 43061374303DNAHomo sapiens 137ggcggcggct ggaggagagc gcggtggaga gccgagcggg cgggcggcgg gtgcggagcg 60ggcgagggag cgcgcgcggc cgccacaaag ctcgggcgcc gcggggctgc atgcggcgta 120cctggcccgg cgcggcgact gctctccggg ctggcggggg ccggccgcga gccccggggg 180ccccgaggcc gcagcttgcc tgcgcgctct gagccttcgc aactcgcgag caaagtttgg 240tggaggcaac gccaagcctg agtcctttct tcctctcgtt ccccaaatcc gagggcagcc 300cgcgggcgtc atgcccgcgc tcctccgcag cctggggtac gcgtgaagcc cgggaggctt 360ggcgccggcg aagacccaag gaccactctt ctgcgtttgg agttgctccc cgcaaccccg 420ggctcgtcgc tttctccatc ccgacccacg cggggcgcgg ggacaacaca ggtcgcggag 480gagcgttgcc attcaagtga ctgcagcagc agcggcagcg cctcggttcc tgagcccacc 540gcaggctgaa ggcattgcgc gtagtccatg cccgtagagg aagtgtgcag atgggattaa 600cgtccacatg gagatatgga agaggaccgg ggattggtac cgtaaccatg gtcagctggg 660gtcgtttcat ctgcctggtc gtggtcacca tggcaacctt gtccctggcc cggccctcct 720tcagtttagt tgaggatacc acattagagc cagaaggagc accatactgg accaacacag 780aaaagatgga aaagcggctc catgctgtgc ctgcggccaa cactgtcaag tttcgctgcc 840cagccggggg gaacccaatg ccaaccatgc ggtggctgaa aaacgggaag gagtttaagc 900aggagcatcg cattggaggc tacaaggtac gaaaccagca ctggagcctc attatggaaa 960gtgtggtccc atctgacaag ggaaattata cctgtgtagt ggagaatgaa tacgggtcca 1020tcaatcacac gtaccacctg gatgttgtgg agcgatcgcc tcaccggccc atcctccaag 1080ccggactgcc ggcaaatgcc tccacagtgg tcggaggaga cgtagagttt gtctgcaagg 1140tttacagtga tgcccagccc cacatccagt ggatcaagca cgtggaaaag aacggcagta 1200aatacgggcc cgacgggctg ccctacctca aggttctcaa ggccgccggt gttaacacca 1260cggacaaaga gattgaggtt ctctatattc ggaatgtaac ttttgaggac gctggggaat 1320atacgtgctt ggcgggtaat tctattggga tatcctttca ctctgcatgg ttgacagttc 1380tgccagcgcc tggaagagaa aaggagatta cagcttcccc agactacctg gagatagcca 1440tttactgcat aggggtcttc ttaatcgcct gtatggtggt aacagtcatc ctgtgccgaa 1500tgaagaacac gaccaagaag ccagacttca gcagccagcc ggctgtgcac aagctgacca 1560aacgtatccc cctgcggaga caggtttcgg ctgagtccag ctcctccatg aactccaaca 1620ccccgctggt gaggataaca acacgcctct cttcaacggc agacaccccc atgctggcag 1680gggtctccga gtatgaactt ccagaggacc caaaatggga gtttccaaga gataagctga 1740cactgggcaa gcccctggga gaaggttgct ttgggcaagt ggtcatggcg gaagcagtgg 1800gaattgacaa agacaagccc aaggaggcgg tcaccgtggc cgtgaagatg ttgaaagatg 1860atgccacaga gaaagacctt tctgatctgg tgtcagagat ggagatgatg aagatgattg 1920ggaaacacaa gaatatcata aatcttcttg gagcctgcac acaggatggg cctctctatg 1980tcatagttga gtatgcctct aaaggcaacc tccgagaata cctccgagcc cggaggccac 2040ccgggatgga gtactcctat gacattaacc gtgttcctga ggagcagatg accttcaagg 2100acttggtgtc atgcacctac cagctggcca gaggcatgga gtacttggct tcccaaaaat 2160gtattcatcg agatttagca gccagaaatg ttttggtaac agaaaacaat gtgatgaaaa 2220tagcagactt tggactcgcc agagatatca acaatataga ctattacaaa aagaccacca 2280atgggcggct tccagtcaag tggatggctc cagaagccct gtttgataga gtatacactc 2340atcagagtga tgtctggtcc ttcggggtgt taatgtggga gatcttcact ttagggggct 2400cgccctaccc agggattccc gtggaggaac tttttaagct gctgaaggaa ggacacagaa 2460tggataagcc agccaactgc accaacgaac tgtacatgat gatgagggac tgttggcatg 2520cagtgccctc ccagagacca acgttcaagc agttggtaga agacttggat cgaattctca 2580ctctcacaac caatgaggaa tacttggacc tcagccaacc tctcgaacag tattcaccta 2640gttaccctga cacaagaagt tcttgttctt caggagatga ttctgttttt tctccagacc 2700ccatgcctta cgaaccatgc cttcctcagt atccacacat aaacggcagt gttaaaacat 2760gaatgactgt gtctgcctgt ccccaaacag gacagcactg ggaacctagc tacactgagc 2820agggagacca tgcctcccag agcttgttgt ctccacttgt atatatggat cagaggagta 2880aataattgga aaagtaatca gcatatgtgt aaagatttat acagttgaaa acttgtaatc 2940ttccccagga ggagaagaag gtttctggag cagtggactg ccacaagcca ccatgtaacc 3000cctctcacct gccgtgcgta ctggctgtgg accagtagga ctcaaggtgg acgtgcgttc 3060tgccttcctt gttaattttg taataattgg agaagattta tgtcagcaca cacttacaga 3120gcacaaatgc agtatatagg tgctggatgt atgtaaatat attcaaatta tgtataaata 3180tatattatat atttacaagg agttattttt tgtattgatt ttaaatggat gtcccaatgc 3240acctagaaaa ttggtctctc tttttttaat agctatttgc taaatgctgt tcttacacat 3300aatttcttaa ttttcaccga gcagaggtgg aaaaatactt ttgctttcag ggaaaatggt 3360ataacgttaa tttattaata aattggtaat atacaaaaca attaatcatt tatagttttt 3420tttgtaattt aagtggcatt tctatgcagg cagcacagca gactagttaa tctattgctt 3480ggacttaact agttatcaga tcctttgaaa agagaatatt tacaatatat gactaatttg 3540gggaaaatga agttttgatt tatttgtgtt taaatgctgc tgtcagacga ttgttcttag 3600acctcctaaa tgccccatat taaaagaact cattcatagg aaggtgtttc attttggtgt 3660gcaaccctgt cattacgtca acgcaacgtc taactggact tcccaagata aatggtacca 3720gcgtcctctt aaaagatgcc ttaatccatt ccttgaggac agaccttagt tgaaatgata 3780gcagaatgtg cttctctctg gcagctggcc ttctgcttct gagttgcaca ttaatcagat 3840tagcctgtat tctcttcagt gaattttgat aatggcttcc agactctttg gcgttggaga 3900cgcctgttag gatcttcaag tcccatcata gaaaattgaa acacagagtt gttctgctga 3960tagttttggg gatacgtcca tctttttaag ggattgcttt catctaattc tggcaggacc 4020tcaccaaaag atccagcctc atacctacat cagacaaaat atcgccgttg ttccttctgt 4080actaaagtat tgtgttttgc tttggaaaca cccactcact ttgcaatagc cgtgcaagat 4140gaatgcagat tacactgatc ttatgtgtta caaaattgga gaaagtattt aataaaacct 4200gttaattttt atactgacaa taaaaatgtt tctacagata ttaatgttaa caagacaaaa 4260taaatgtcac gcaacttatt tttttaataa aaaaaaaaaa aaa 43031383011DNAHomo sapiens 138ggcggcggct ggaggagagc gcggtggaga gccgagcggg cgggcggcgg gtgcggagcg 60ggcgagggag cgcgcgcggc cgccacaaag ctcgggcgcc gcggggctgc atgcggcgta 120cctggcccgg cgcggcgact gctctccggg ctggcggggg ccggccgcga gccccggggg 180ccccgaggcc gcagcttgcc tgcgcgctct gagccttcgc aactcgcgag caaagtttgg 240tggaggcaac gccaagcctg agtcctttct tcctctcgtt ccccaaatcc gagggcagcc 300cgcgggcgtc atgcccgcgc tcctccgcag cctggggtac gcgtgaagcc cgggaggctt 360ggcgccggcg aagacccaag gaccactctt ctgcgtttgg agttgctccc cgcaaccccg 420ggctcgtcgc tttctccatc ccgacccacg cggggcgcgg ggacaacaca ggtcgcggag 480gagcgttgcc attcaagtga ctgcagcagc agcggcagcg cctcggttcc tgagcccacc 540gcaggctgaa ggcattgcgc gtagtccatg cccgtagagg aagtgtgcag atgggattaa 600cgtccacatg gagatatgga agaggaccgg ggattggtac cgtaaccatg gtcagctggg 660gtcgtttcat ctgcctggtc gtggtcacca tggcaacctt gtccctggcc cggccctcct 720tcagtttagt tgaggatacc acattagagc cagaagatgc catctcatcc ggagatgatg 780aggatgacac cgatggtgcg gaagattttg tcagtgagaa cagtaacaac aagagagcac 840catactggac caacacagaa aagatggaaa agcggctcca tgctgtgcct gcggccaaca 900ctgtcaagtt tcgctgccca gccgggggga acccaatgcc aaccatgcgg tggctgaaaa 960acgggaagga gtttaagcag gagcatcgca ttggaggcta caaggtacga aaccagcact 1020ggagcctcat tatggaaagt gtggtcccat ctgacaaggg aaattatacc tgtgtagtgg 1080agaatgaata cgggtccatc aatcacacgt accacctgga tgttgtggag cgatcgcctc 1140accggcccat cctccaagcc ggactgccgg caaatgcctc cacagtggtc ggaggagacg 1200tagagtttgt ctgcaaggtt tacagtgatg cccagcccca catccagtgg atcaagcacg 1260tggaaaagaa cggcagtaaa tacgggcccg acgggctgcc ctacctcaag gttctcaagc 1320actcggggat aaatagttcc aatgcagaag tgctggctct gttcaatgtg accgaggcgg 1380atgctgggga atatatatgt aaggtctcca attatatagg gcaggccaac cagtctgcct 1440ggctcactgt cctgccaaaa cagcaagcgc ctggaagaga aaaggagatt acagcttccc 1500cagactacct ggagatagcc atttactgca taggggtctt cttaatcgcc tgtatggtgg 1560taacagtcat cctgtgccga atgaagaaca cgaccaagaa gccagacttc agcagccagc 1620cggctgtgca caagctgacc aaacgtatcc ccctgcggag acaggtaaca gtttcggctg 1680agtccagctc ctccatgaac tccaacaccc cgctggtgag gataacaaca cgcctctctt 1740caacggcaga cacccccatg ctggcagggg tctccgagta tgaacttcca gaggacccaa 1800aatgggagtt tccaagagat aagctgacac tgggcaagcc cctgggagaa ggttgctttg 1860ggcaagtggt catggcggaa gcagtgggaa ttgacaaaga caagcccaag gaggcggtca 1920ccgtggccgt gaagatgttg aaagatgatg ccacagagaa agacctttct gatctggtgt 1980cagagatgga gatgatgaag atgattggga aacacaagaa tatcataaat cttcttggag 2040cctgcacaca ggatgggcct ctctatgtca tagttgagta tgcctctaaa ggcaacctcc 2100gagaatacct ccgagcccgg aggccacccg ggatggagta ctcctatgac attaaccgtg 2160ttcctgagga gcagatgacc ttcaaggact tggtgtcatg cacctaccag ctggccagag 2220gcatggagta cttggcttcc caaaaatgta ttcatcgaga tttagcagcc agaaatgttt 2280tggtaacaga aaacaatgtg atgaaaatag cagactttgg actcgccaga gatatcaaca 2340atatagacta ttacaaaaag accaccaatg ggcggcttcc agtcaagtgg atggctccag 2400aagccctgtt tgatagagta tacactcatc agagtgatgt ctggtccttc ggggtgttaa 2460tgtgggagat cttcacttta gggggctcgc cctacccagg gattcccgtg gaggaacttt 2520ttaagctgct gaaggaagga cacagaatgg ataagccagc caactgcacc aacgaactgt 2580acatgatgat gagggactgt tggcatgcag tgccctccca gagaccaacg ttcaagcagt 2640tggtagaaga cttggatcga attctcactc tcacaaccaa tgagatctga aagtttatgg 2700cttcattgag aaactgggaa aagttggtca ggcgcagtgg ctcatgcctg taatcccagc 2760actttgggag gccgaggcag gcggatcatg aggtcaggag ttccagacca gcctggccaa 2820catggtgaaa ccctgtctct actaaagata caaaaaatta gccgggcgtg ttggtgtgca 2880cctgtaatcc cagctactcc gggaggctga ggcaggagag tcacttgaac cggggaggcg 2940gaggttgcag tgagccgaga tcatgccatt gcattccagc cttggcgaca gagcgagact 3000ccgtctcaaa a 30111394286DNAHomo sapiens 139gtcgcgggca gctggcgccg cgcggtcctg ctctgccggt cgcacggacg caccggcggg 60ccgccggccg gagggacggg gcgggagctg ggcccgcgga cagcgagccg gagcgggagc 120cgcgcgtagc gagccgggct ccggcgctcg ccagtctccc gagcggcgcc cgcctcccgc 180cggtgcccgc gccgggccgt ggggggcagc atgcccgcgc gcgctgcctg aggacgccgc 240ggcccccgcc cccgccatgg gcgcccctgc ctgcgccctc gcgctctgcg tggccgtggc 300catcgtggcc ggcgcctcct cggagtcctt ggggacggag cagcgcgtcg tggggcgagc 360ggcagaagtc ccgggcccag agcccggcca gcaggagcag ttggtcttcg gcagcgggga 420tgctgtggag ctgagctgtc ccccgcccgg gggtggtccc atggggccca ctgtctgggt 480caaggatggc acagggctgg tgccctcgga gcgtgtcctg gtggggcccc agcggctgca 540ggtgctgaat gcctcccacg aggactccgg ggcctacagc tgccggcagc ggctcacgca 600gcgcgtactg tgccacttca gtgtgcgggt gacagacgct ccatcctcgg gagatgacga 660agacggggag gacgaggctg aggacacagg tgtggacaca ggggcccctt actggacacg 720gcccgagcgg atggacaaga agctgctggc cgtgccggcc gccaacaccg tccgcttccg 780ctgcccagcc gctggcaacc ccactccctc catctcctgg ctgaagaacg gcagggagtt 840ccgcggcgag caccgcattg gaggcatcaa gctgcggcat cagcagtgga gcctggtcat 900ggaaagcgtg gtgccctcgg accgcggcaa ctacacctgc gtcgtggaga acaagtttgg 960cagcatccgg cagacgtaca cgctggacgt gctggagcgc tccccgcacc ggcccatcct 1020gcaggcgggg ctgccggcca accagacggc ggtgctgggc agcgacgtgg agttccactg 1080caaggtgtac agtgacgcac agccccacat ccagtggctc aagcacgtgg aggtgaatgg 1140cagcaaggtg ggcccggacg gcacacccta cgttaccgtg ctcaagacgg cgggcgctaa 1200caccaccgac aaggagctag aggttctctc cttgcacaac gtcacctttg aggacgccgg 1260ggagtacacc tgcctggcgg gcaattctat tgggttttct catcactctg cgtggctggt 1320ggtgctgcca gccgaggagg agctggtgga ggctgacgag gcgggcagtg tgtatgcagg 1380catcctcagc tacggggtgg gcttcttcct gttcatcctg gtggtggcgg ctgtgacgct 1440ctgccgcctg cgcagccccc ccaagaaagg cctgggctcc cccaccgtgc acaagatctc 1500ccgcttcccg ctcaagcgac

aggtgtccct ggagtccaac gcgtccatga gctccaacac 1560accactggtg cgcatcgcaa ggctgtcctc aggggagggc cccacgctgg ccaatgtctc 1620cgagctcgag ctgcctgccg accccaaatg ggagctgtct cgggcccggc tgaccctggg 1680caagcccctt ggggagggct gcttcggcca ggtggtcatg gcggaggcca tcggcattga 1740caaggaccgg gccgccaagc ctgtcaccgt agccgtgaag atgctgaaag acgatgccac 1800tgacaaggac ctgtcggacc tggtgtctga gatggagatg atgaagatga tcgggaaaca 1860caaaaacatc atcaacctgc tgggcgcctg cacgcagggc gggcccctgt acgtgctggt 1920ggagtacgcg gccaagggta acctgcggga gtttctgcgg gcgcggcggc ccccgggcct 1980ggactactcc ttcgacacct gcaagccgcc cgaggagcag ctcaccttca aggacctggt 2040gtcctgtgcc taccaggtgg cccggggcat ggagtacttg gcctcccaga agtgcatcca 2100cagggacctg gctgcccgca atgtgctggt gaccgaggac aacgtgatga agatcgcaga 2160cttcgggctg gcccgggacg tgcacaacct cgactactac aagaagacga ccaacggccg 2220gctgcccgtg aagtggatgg cgcctgaggc cttgtttgac cgagtctaca ctcaccagag 2280tgacgtctgg tcctttgggg tcctgctctg ggagatcttc acgctggggg gctccccgta 2340ccccggcatc cctgtggagg agctcttcaa gctgctgaag gagggccacc gcatggacaa 2400gcccgccaac tgcacacacg acctgtacat gatcatgcgg gagtgctggc atgccgcgcc 2460ctcccagagg cccaccttca agcagctggt ggaggacctg gaccgtgtcc ttaccgtgac 2520gtccaccgac gagtacctgg acctgtcggc gcctttcgag cagtactccc cgggtggcca 2580ggacaccccc agctccagct cctcagggga cgactccgtg tttgcccacg acctgctgcc 2640cccggcccca cccagcagtg ggggctcgcg gacgtgaagg gccactggtc cccaacaatg 2700tgaggggtcc ctagcagccc accctgctgc tggtgcacag ccactccccg gcatgagact 2760cagtgcagat ggagagacag ctacacagag ctttggtctg tgtgtgtgtg tgtgcgtgtg 2820tgtgtgtgtg tgtgcacatc cgcgtgtgcc tgtgtgcgtg cgcatcttgc ctccaggtgc 2880agaggtaccc tgggtgtccc cgctgctgtg caacggtctc ctgactggtg ctgcagcacc 2940gaggggcctt tgttctgggg ggacccagtg cagaatgtaa gtgggcccac ccggtgggac 3000ccccgtgggg cagggagctg ggcccgacat ggctccggcc tctgcctttg caccacggga 3060catcacaggg tgggcctcgg cccctcccac acccaaagct gagcctgcag ggaagcccca 3120catgtccagc accttgtgcc tggggtgtta gtggcaccgc ctccccacct ccaggctttc 3180ccacttccca ccctgcccct cagagactga aattacgggt acctgaagat gggagccttt 3240accttttatg caaaaggttt attccggaaa ctagtgtaca tttctataaa tagatgctgt 3300gtatatggta tatatacata tatatatata acatatatgg aagaggaaaa ggctggtaca 3360acggaggcct gcgaccctgg gggcacagga ggcaggcatg gccctgggcg gggcgtgggg 3420gggcgtggag ggaggcccca gggggtctca cccatgcaag cagaggacca gggccttttc 3480tggcaccgca gttttgtttt aaaactggac ctgtatattt gtaaagctat ttatgggccc 3540ctggcactct tgttcccaca ccccaacact tccagcattt agctggccac atggcggaga 3600gttttaattt ttaacttatt gacaaccgag aaggtttatc ccgccgatag agggacggcc 3660aagaatgtac gtccagcctg ccccggagct ggaggatccc ctccaagcct aaaaggttgt 3720taatagttgg aggtgattcc agtgaagata ttttatttcc tttgtccttt ttcaggagaa 3780ttagatttct ataggatttt tctttaggag atttattttt tggacttcaa agcaagctgg 3840tattttcata caaattcttc taattgctgt gtgtcccagg cagggagacg gtttccaggg 3900aggggccggc cctgtgtgca ggttccgatg ttattagatg ttacaagttt atatatatct 3960atatatataa tttattgagt ttttacaaga tgtatttgtt gtagacttaa cacttcttac 4020gcaatgcttc tagagtttta tagcctggac tgctaccttt caaagcttgg agggaagccg 4080tgaattcagt tggttcgttc tgtactgtta ctgggccctg agtctgggca gctgtccctt 4140gcttgcctgc agggccatgg ctcagggtgg tctcttcttg gggcccagtg catggtggcc 4200agaggtgtca cccaaaccgg caggtgcgat tttgttaacc cagcgacgaa ctttccgaaa 4260aataaagaca cctggttgct aacctg 42861403950DNAHomo sapiens 140gtcgcgggca gctggcgccg cgcggtcctg ctctgccggt cgcacggacg caccggcggg 60ccgccggccg gagggacggg gcgggagctg ggcccgcgga cagcgagccg gagcgggagc 120cgcgcgtagc gagccgggct ccggcgctcg ccagtctccc gagcggcgcc cgcctcccgc 180cggtgcccgc gccgggccgt ggggggcagc atgcccgcgc gcgctgcctg aggacgccgc 240ggcccccgcc cccgccatgg gcgcccctgc ctgcgccctc gcgctctgcg tggccgtggc 300catcgtggcc ggcgcctcct cggagtcctt ggggacggag cagcgcgtcg tggggcgagc 360ggcagaagtc ccgggcccag agcccggcca gcaggagcag ttggtcttcg gcagcgggga 420tgctgtggag ctgagctgtc ccccgcccgg gggtggtccc atggggccca ctgtctgggt 480caaggatggc acagggctgg tgccctcgga gcgtgtcctg gtggggcccc agcggctgca 540ggtgctgaat gcctcccacg aggactccgg ggcctacagc tgccggcagc ggctcacgca 600gcgcgtactg tgccacttca gtgtgcgggt gacagacgct ccatcctcgg gagatgacga 660agacggggag gacgaggctg aggacacagg tgtggacaca ggggcccctt actggacacg 720gcccgagcgg atggacaaga agctgctggc cgtgccggcc gccaacaccg tccgcttccg 780ctgcccagcc gctggcaacc ccactccctc catctcctgg ctgaagaacg gcagggagtt 840ccgcggcgag caccgcattg gaggcatcaa gctgcggcat cagcagtgga gcctggtcat 900ggaaagcgtg gtgccctcgg accgcggcaa ctacacctgc gtcgtggaga acaagtttgg 960cagcatccgg cagacgtaca cgctggacgt gctggagcgc tccccgcacc ggcccatcct 1020gcaggcgggg ctgccggcca accagacggc ggtgctgggc agcgacgtgg agttccactg 1080caaggtgtac agtgacgcac agccccacat ccagtggctc aagcacgtgg aggtgaatgg 1140cagcaaggtg ggcccggacg gcacacccta cgttaccgtg ctcaaggtgt ccctggagtc 1200caacgcgtcc atgagctcca acacaccact ggtgcgcatc gcaaggctgt cctcagggga 1260gggccccacg ctggccaatg tctccgagct cgagctgcct gccgacccca aatgggagct 1320gtctcgggcc cggctgaccc tgggcaagcc ccttggggag ggctgcttcg gccaggtggt 1380catggcggag gccatcggca ttgacaagga ccgggccgcc aagcctgtca ccgtagccgt 1440gaagatgctg aaagacgatg ccactgacaa ggacctgtcg gacctggtgt ctgagatgga 1500gatgatgaag atgatcggga aacacaaaaa catcatcaac ctgctgggcg cctgcacgca 1560gggcgggccc ctgtacgtgc tggtggagta cgcggccaag ggtaacctgc gggagtttct 1620gcgggcgcgg cggcccccgg gcctggacta ctccttcgac acctgcaagc cgcccgagga 1680gcagctcacc ttcaaggacc tggtgtcctg tgcctaccag gtggcccggg gcatggagta 1740cttggcctcc cagaagtgca tccacaggga cctggctgcc cgcaatgtgc tggtgaccga 1800ggacaacgtg atgaagatcg cagacttcgg gctggcccgg gacgtgcaca acctcgacta 1860ctacaagaag acgaccaacg gccggctgcc cgtgaagtgg atggcgcctg aggccttgtt 1920tgaccgagtc tacactcacc agagtgacgt ctggtccttt ggggtcctgc tctgggagat 1980cttcacgctg gggggctccc cgtaccccgg catccctgtg gaggagctct tcaagctgct 2040gaaggagggc caccgcatgg acaagcccgc caactgcaca cacgacctgt acatgatcat 2100gcgggagtgc tggcatgccg cgccctccca gaggcccacc ttcaagcagc tggtggagga 2160cctggaccgt gtccttaccg tgacgtccac cgacgagtac ctggacctgt cggcgccttt 2220cgagcagtac tccccgggtg gccaggacac ccccagctcc agctcctcag gggacgactc 2280cgtgtttgcc cacgacctgc tgcccccggc cccacccagc agtgggggct cgcggacgtg 2340aagggccact ggtccccaac aatgtgaggg gtccctagca gcccaccctg ctgctggtgc 2400acagccactc cccggcatga gactcagtgc agatggagag acagctacac agagctttgg 2460tctgtgtgtg tgtgtgtgcg tgtgtgtgtg tgtgtgtgca catccgcgtg tgcctgtgtg 2520cgtgcgcatc ttgcctccag gtgcagaggt accctgggtg tccccgctgc tgtgcaacgg 2580tctcctgact ggtgctgcag caccgagggg cctttgttct ggggggaccc agtgcagaat 2640gtaagtgggc ccacccggtg ggacccccgt ggggcaggga gctgggcccg acatggctcc 2700ggcctctgcc tttgcaccac gggacatcac agggtgggcc tcggcccctc ccacacccaa 2760agctgagcct gcagggaagc cccacatgtc cagcaccttg tgcctggggt gttagtggca 2820ccgcctcccc acctccaggc tttcccactt cccaccctgc ccctcagaga ctgaaattac 2880gggtacctga agatgggagc ctttaccttt tatgcaaaag gtttattccg gaaactagtg 2940tacatttcta taaatagatg ctgtgtatat ggtatatata catatatata tataacatat 3000atggaagagg aaaaggctgg tacaacggag gcctgcgacc ctgggggcac aggaggcagg 3060catggccctg ggcggggcgt gggggggcgt ggagggaggc cccagggggt ctcacccatg 3120caagcagagg accagggcct tttctggcac cgcagttttg ttttaaaact ggacctgtat 3180atttgtaaag ctatttatgg gcccctggca ctcttgttcc cacaccccaa cacttccagc 3240atttagctgg ccacatggcg gagagtttta atttttaact tattgacaac cgagaaggtt 3300tatcccgccg atagagggac ggccaagaat gtacgtccag cctgccccgg agctggagga 3360tcccctccaa gcctaaaagg ttgttaatag ttggaggtga ttccagtgaa gatattttat 3420ttcctttgtc ctttttcagg agaattagat ttctatagga tttttcttta ggagatttat 3480tttttggact tcaaagcaag ctggtatttt catacaaatt cttctaattg ctgtgtgtcc 3540caggcaggga gacggtttcc agggaggggc cggccctgtg tgcaggttcc gatgttatta 3600gatgttacaa gtttatatat atctatatat ataatttatt gagtttttac aagatgtatt 3660tgttgtagac ttaacacttc ttacgcaatg cttctagagt tttatagcct ggactgctac 3720ctttcaaagc ttggagggaa gccgtgaatt cagttggttc gttctgtact gttactgggc 3780cctgagtctg ggcagctgtc ccttgcttgc ctgcagggcc atggctcagg gtggtctctt 3840cttggggccc agtgcatggt ggccagaggt gtcacccaaa ccggcaggtg cgattttgtt 3900aacccagcga cgaactttcc gaaaaataaa gacacctggt tgctaacctg 39501412085DNAHomo sapiens 141atgggcgccc ctgcctgcgc cctcgcgctc tgcgtggccg tggccatcgt ggccggcgcc 60tcctcggagt ccttggggac ggagcagcgc gtcgtggggc gagcggcaga agtcccgggc 120ccagagcccg gccagcagga gcagttggtc ttcggcagcg gggatgctgt ggagctgagc 180tgtcccccgc ccgggggtgg tcccatgggg cccactgtct gggtcaagga tggcacaggg 240ctggtgccct cggagcgtgt cctggtgggg ccccagcggc tgcaggtgct gaatgcctcc 300cacgaggact ccggggccta cagctgccgg cagcggctca cgcagcgcgt actgtgccac 360ttcagtgtgc gggtgacaga cgctccatcc tcgggagatg acgaagacgg ggaggacgag 420gctgaggaca caggtgtgga cacaggggcc ccttactgga cacggcccga gcggatggac 480aagaagctgc tggccgtgcc ggccgccaac accgtccgct tccgctgccc agccgctggc 540aaccccactc cctccatctc ctggctgaag aacggcaggg agttccgcgg cgagcaccgc 600attggaggca tcaagctgcg gcatcagcag tggagcctgg tcatggaaag cgtggtgccc 660tcggaccgcg gcaactacac ctgcgtcgtg gagaacaagt ttggcagcat ccggcagacg 720tacacgctgg acgtgctgga gcgctccccg caccggccca tcctgcaggc ggggctgccg 780gccaaccaga cggcggtgct gggcagcgac gtggagttcc actgcaaggt gtacagtgac 840gcacagcccc acatccagtg gctcaagcac gtggaggtga acggcagcaa ggtgggcccg 900gacggcacac cctacgttac cgtgctcaag gtgtccctgg agtccaacgc gtccatgagc 960tccaacacac cactggtgcg catcgcaagg ctgtcctcag gggagggccc cacgctggcc 1020aatgtctccg agctcgagct gcctgccgac cccaaatggg agctgtctcg ggcccggctg 1080accctgggca agccccttgg ggagggctgc ttcggccagg tggtcatggc ggaggccatc 1140ggcattgaca aggaccgggc cgccaagcct gtcaccgtag ccgtgaagat gctgaaagac 1200gatgccactg acaaggacct gtcggacctg gtgtctgaga tggagatgat gaagatgatc 1260gggaaacaca aaaacatcat caacctgctg ggcgcctgca cgcagggcgg gcccctgtac 1320gtgctggtgg agtacgcggc caagggtaac ctgcgggagt ttctgcgggc gcggcggccc 1380ccgggcctgg actactcctt cgacacctgc aagccgcccg aggagcagct caccttcaag 1440gacctggtgt cctgtgccta ccaggtggcc cggggcatgg agtacttggc ctcccagaag 1500tgcatccaca gggacctggc tgcccgcaat gtgctggtga ccgaggacaa cgtgatgaag 1560atcgcagact tcgggctggc ccgggacgtg cacaacctcg actactacaa gaagacaacc 1620aacggccggc tgcccgtgaa gtggatggcg cctgaggcct tgtttgaccg agtctacact 1680caccagagtg acgtctggtc ctttggggtc ctgctctggg agatcttcac gctggggggc 1740tccccgtacc ccggcatccc tgtggaggag ctcttcaagc tgctgaagga gggccaccgc 1800atggacaagc ccgccaactg cacacacgac ctgtacatga tcatgcggga gtgctggcat 1860gccgcgccct cccagaggcc caccttcaag cagctggtgg aggacctgga ccgtgtcctt 1920accgtgacgt ccaccgacga gtacctggac ctgtcggcgc ctttcgagca gtactccccg 1980ggtggccagg acacccccag ctccagctcc tcaggggacg actccgtgtt tgcccacgac 2040ctgctgcccc cggccccacc cagcagtggg ggctcgcgga cgtga 20851424431DNAHomo sapiens 142aggcggggct ggagtggtgg aaggggggtg gcaggtctgc attgccgctt ccctggtgcc 60gggagcagtc gccgctgccg cctccgcccg cggccgggac ccccgtcctc gcccgggact 120ccttacccgg ggaacctaga ccaggtctcc agaggcttgt ggaagagaag caggcgaccc 180ttcctgagtt atcctggctt agcctcccaa tctggctccc cttccccttc ccattcccct 240gctccccctg tcccttcccc atccacccaa ctgaactggg tataggtcaa agctcctctc 300tttccttttc cttcctaggc actcattggc taggacctgt ttgctctttt ttttgtgccc 360agagatactg gaacacgctt catctaagta actgtgggga ggggtctttt tgactctaca 420agtccttgag caaaaagctg aaaaagaagc aggaggtgga gaagacccag tgaagtgccc 480caagccccat catggaagag ggcttccgag accgggcagc tttcatccgt ggggccaaag 540acattgctaa ggaagtcaaa aagcatgcgg ccaagaaggt ggtgaagggc ctggacagag 600tccaggacga atattcccga agatcgtact cccgctttga ggaggaggat gatgatgatg 660acttccctgc tcccagtgat ggttattacc gaggagaagg gacccaggat gaggaggaag 720gtggtgcatc cagtgatgct actgagggcc atgacgagga tgatgagatc tatgaagggg 780aatatcaggg cattccccgg gcagagtctg ggggcaaagg cgagcggatg gcagatgggg 840cgcccctggc tggagtaagg gggggcttga gtgatgggga gggtccccct gggggccggg 900gggaggcaca acgacggaaa gaacgagaag aactggccca acagtatgaa gccatcctac 960gggagtgtgg ccacggccgc ttccagtgga cactgtattt tgtgcttggt ctggcgctga 1020tggctgacgg tgtggaggtc tttgtggtgg gcttcgtgct gcccagcgct gagaaagaca 1080tgtgcctgtc cgactccaac aaaggcatgc taggcctcat cgtctacctg ggcatgatgg 1140tgggagcctt cctctgggga ggtctggctg accggctggg tcggaggcag tgtctgctca 1200tctcgctctc agtcaacagc gtcttcgcct tcttctcatc ttttgtccag ggttacggca 1260ctttcctctt ctgccgccta ctttctgggg ttgggattgg agggtccatc cccattgtct 1320tctcctattt ctccgagttt ctggcccagg agaaacgagg ggagcatttg agctggctct 1380gcatgttttg gatgattggt ggcgtgtacg cagctgctat ggcctgggcc atcatccccc 1440actatgggtg gagttttcag atgggttctg cctaccagtt ccacagctgg agggtcttcg 1500tcctcgtctg cgcctttcct tctgtgtttg ccattggggc tctgaccacg cagcctgaga 1560gcccccgttt cttcctagag aatggaaagc atgatgaggc ctggatggtg ctgaagcagg 1620tccatgatac caacatgcga gccaaaggac atcctgagcg agtgttctca gtaacccaca 1680ttaagacgat tcatcaggag gatgaattga ttgagatcca gtcggacaca gggacctggt 1740accagcgctg gggggtccgg gccttgagcc taggggggca ggtttggggg aattttctct 1800cctgttttgg tcccgaatat cggcgcatca ctctgatgat gatgggtgtg tggttcacca 1860tgtcattcag ctactatggc ctgaccgtct ggtttcctga catgatccgc catctccagg 1920cagtggacta cgcatcccgc accaaagtgt tccccgggga gcgcgtagag catgtaactt 1980ttaacttcac gttggagaat cagatccacc gaggcgggca gtacttcaat gacaagttca 2040ttgggctgcg gctcaagtca gtgtcctttg aggattccct gtttgaagag tgttattttg 2100aggatgtcac atccagcaac acgtttttcc gcaactgcac attcatcaac actgtgttct 2160ataacactga cctgttcgag tacaagtttg tgaacagccg tctgataaac agtacattcc 2220tgcacaacaa ggagggctgc ccgctagacg tgacagggac gggcgaaggt gcctacatgg 2280tatactttgt gagcttcctg gggacactgg cagtgcttcc tgggaatatc gtgtctgccc 2340tgctcatgga caagatcggc aggctcagaa tgcttgctgg ctccagcgtg atgtcctgtg 2400tctcctgctt cttcctgtct tttgggaaca gtgagtcggc catgatcgct ctgctctgcc 2460tttttggcgg ggtcagcatt gcatcctgga atgcgctgga cgtgttgact gttgaactct 2520acccctcaga caagaggacc acagcttttg gcttcctgaa tgccctgtgt aagctggcag 2580ctgtgctggg gatcagcatc ttcacatcct tcgtgggaat caccaaggct gcacccatcc 2640tctttgcctc agctgccctt gcccttggca gctctctggc cctgaagctg cctgagaccc 2700gggggcaggt gctgcagtga aggggtctct agggctttgg gattggcagg cacactgtga 2760gaccaacaac tccttccttc ccctccctgc cctgccatcc tgacctccag agccctcact 2820ccccactccc cgtgtttggt gtcttagctg tgtgtgcgtg tgcgtgtgca tgtgtgtaaa 2880ccccgtgggc agggactaca gggaaggctc cttcatccca gttttgagat gaagctgtac 2940tccccatttc ccactgccct tgactttgca caagagaagg ctgagcccca tccttctccc 3000cctgttagag aggggccctt gcttccctgt tccaggggtt ccagaatagg cttcctgcct 3060tccccatcat tccctctgcc taggccctgg tgaaaccaca ggtatgcaat tatgctaggg 3120gctggggctc tggtgtagac catggaccaa aagaacttct tagagtctga agagtgggcc 3180tcgggtgccc tctcacatct cctgttggat gctgggggag aagcaataaa cctcagccct 3240ctggcctcca ctttcctctc aatttgggct gcaaatatga agcctgaatt ttatgaaatt 3300agctttctga ttcttattta ttaatagatt aagttctgag gcagctccgc aggactgtgt 3360gtgaatgtgt atgtatactt acatatgtgt gtgcatgtgc catggggcgg ggggtatcac 3420tatactgtcc tcaaatataa gccaagggta atttcagcgg atgcacacac aaccctgcct 3480cccacagttc ctcccctaat ctggtttctg tgttgagcct gggatggagg agccctaggc 3540cagcctggga taagagtccc acagtctagg gagatctgag ggcatccgac aaggcccatc 3600tccttccctc ctcaagaagc agaggcctcc tctggagtga gaggctccac ccactacagc 3660acaggcggga atagcacagc tgccctccca tgctccctac ctgtcccctc acagggaggg 3720gagcagggga gggaaagaaa ccaggcatct ggtcaaacca gcagatcaaa aagcacaaag 3780agctggggca gaggcaggaa gcaggggccc tcctggcagc tcctctgagt ggggagaggt 3840tgggcagtga gtgagggacc cctaatgcag ggactagaag cctcagtttc cccattttac 3900ccttccacac aatagcctct gtaggttagg ctgccccatc ccaccctact ctgtgtggct 3960gctttctttg gtgccctccc ctcaccccac tgtagctgtg acgtgttgta gtttttagat 4020gtttgtaaaa tgtttaaaaa aatgttaaaa ggaaaaaagt gaaaataaca aaaaagaaaa 4080tcaaaattca ccttcgtcat gctgcgtcca gtgccccaac cctgtggtca ctctccccat 4140tttgtaacac tgtaccaggt ggtgactgtt taactctttg gtgtctgtgc tcaaaagact 4200gccttctcca gtgcccagtg tatgagtgtg tgccctgtgc ccttgtccct cactccccac 4260atgctggacg tagccctctt cctcgcaccc ctgggaggga cccatccatc tcccttgctc 4320tcctggggaa ccctaaaccc aactctgttg atgtgaaaaa tgcagtgaaa aatattgacg 4380aaaaataaaa cggaaacaaa tcctcaaaat acaaaaaaaa aaaaaaaaaa a 44311435311DNAHomo sapiens 143agcataacct tcggtggcag gacaaatcag gccagcacgc agtctgccaa gtcctgctcg 60ctccctgtca agaaaaacag ctggatccat ttctaatcaa cacttcccaa cgcaacactt 120ctgagtctct gaaggagacc agagcttgaa actttccaga cttccaacag acatcgagtg 180caaaaggata tttaggttgt ctttgcacaa atctggttga tttgagagat aaaggggggg 240ggaaccagtg tgactttcac ctaagaagtc acatgaacat atttcacatt tgaactacat 300aatgaatgat ggttattgaa atagcccaaa cctctaccac agagcgaggg atatagctca 360aggggcaacc aggcagtcgc agaaccaagg aatggatgac tacaagtatc aggacaatta 420tgggggctat gctcccagtg atggctatta ccgcggcaat gagtccaacc cagaagaaga 480tgcacagagt gatgtcaccg aaggccatga tgaggaagac gagatctatg agggcgagta 540ccagggtatc cctcacccag atgatgtcaa ggccaagcag gccaagatgg cgccctccag 600aatggacagc cttcggggcc agacagacct gatggctgag aggctggaag atgaggagca 660gttggcccac cagtacgaga ccatcatgga tgagtgtggc catggccgct tccagtggat 720cctctttttc gtcttgggtt tggccctgat ggccgatggg gtggaagtgt tcgtggtgag 780ttttgccctg cccagtgcag agaaggacat gtgtctgtcc agttccaaaa aaggaatgct 840agggatgata gtctacttgg gaatgatggc gggcgccttc atcctgggag gcctggctga 900taagctggga aggaagcgag tcctcagcat gtctctggcc gtcaatgcct ccttcgcctc 960cctctcttcc ttcgtgcagg gatatggagc cttcctcttc tgccgactca tctcaggcat 1020cggtattggg ggtgctctac cgattgtttt tgcctatttt tctgaattct tgtctcggga 1080gaagcgagga gaacacctca gttggctggg catcttctgg atgactgggg gcctgtacgc 1140atctgccatg gcctggagca tcatcccaca ctatggctgg ggcttcagca tggggaccaa 1200ttaccacttc catagctgga gagtgtttgt catcgtctgt gctctgccct gcaccgtgtc 1260catggtggcc ctgaagttca tgccagagag cccaaggttt ctgctagaga tgggcaaaca 1320tgatgaagcc tggatgattc tcaagcaagt ccatgacacc aacatgagag ctaaggggac 1380cccagagaaa gtgttcacgg tttccaacat caaaactccc aagcaaatgg atgaattcat 1440tgagatccaa agttcaacag gaacctggta ccagcgctgg ctggtcagat tcaagaccat 1500tttcaagcag gtctgggata atgccctgta ctgtgtgatg gggccctaca gaatgaatac 1560actgattctg gccgtggttt ggtttgccat ggcattcagt tactatggac tgacagtttg 1620gtttcctgat atgatccgct attttcaaga tgaagaatac

aagtctaaaa tgaaggtgtt 1680ttttggtgag catgtgtacg gcgccacaat caacttcacg atggaaaatc agatccacca 1740acatgggaaa cttgtgaatg ataagttcac aagaatgtac tttaaacatg tactctttga 1800ggacacattc tttgacgagt gctattttga agacgtaaca tcaacagata cctacttcaa 1860aaattgtacc attgaatcaa ccatctttta caacacagac ctctacgagc acaagttcat 1920caactgtcgg tttatcaact ccaccttcct ggagcagaag gagggctgcc acatggactt 1980ggagcaagat aatgacttcc tgatttacct cgtcagcttc ctgggcagcc tgtctgtctt 2040acccgggaac atcatttctg ccctgctcat ggatagaatt ggaaggctca agatgattgg 2100tggctccatg ctaatctctg cagtctgctg cttcttcctg ttttttggca acagtgagtc 2160tgcaatgatc ggctggcagt gcctgttctg tgggacaagc attgcagcct ggaatgctct 2220ggatgtgatc acagtggagc tgtatcccac caaccagaga gcaacagcct tcggcattct 2280caatggatta tgcaaatttg gcgccatcct gggaaacacc atctttgctt cttttgttgg 2340gataaccaaa gtggtcccca tccttctggc tgctgcttct ctggttgggg gtggcctgat 2400tgcccttcga ctgccagaga ctcgagaaca ggtcctgatg tgaacaacct atgggaaaag 2460gaaaggtcga gagaatcttg tccaggacac tgaaatgcat ccacacttcc tgcctatcac 2520ggtccggagg acaccttgga tagcacggga ggagaagttg actttgtgac ccctagttta 2580ggacccactt cagctgtcaa tatgtttgta actcaggtga ctgatttggg ggtgccctga 2640gccaccctta gaatcacaga gctgcgtgtt taacttcaag tcttcccagt ccaaggcagg 2700gagaggattc tccagtgagt gcacacacta tgcgaggagc aagcatttct ctaagtcaag 2760tgcaaggact taacttgcgt ttgaaaagga attagagggt cagaaacacc caggttcctc 2820cagaaagctc cttggagccc aacaacttaa caaatcaact tggctggaag ttagagtcat 2880tatatgaaga ttgggcttga agtatatatt tttgcattta aaagtatcac ctatcatatt 2940ttccactcga aaattgacat agtagcattg aggatactct gatctagaaa gccaagtatt 3000tgagcaacat ctatagagat ctacttttct cctatgtctc ctaggctttc catgataatt 3060aggtaataca tttaagaagg atatttattt ctgttttgct ctattcaaag aaacggaatg 3120ggatagttat tctgtaaact aagtttgtat ataactttat ttgggtttaa tttccacaac 3180tggtatctgc aaatattgcc agcattttag ccatattttg ggagaacttg gtgtttgagg 3240tcccaggaaa tgaggtctga tcaaatgaaa tgcaagcaca atttcttaca gccatttaac 3300tttctgttgg gaggatgaat taacaaactc acattgtgca gtctgcttaa tccaggcact 3360tttctttgtg caggtgtagt gagtagttac ttctctccct tacacagatg acttgtgaaa 3420ctcaagctca ccatcttcag tgctggcatt ttactttgcc actacccaaa aacaatgtga 3480gatgtgttca gtggcctctg gtactctttg caggcaagaa tcaaacaaca tggggactga 3540gggaaggatg gggaagtgta gccacagttc ttccaaatgt aaatactttt tgtttgttct 3600agtggtaaaa tgcaaatgca atccatattt gttaggatgg tcaggtctca tgagaaatct 3660atgctatgtg tccagagctt ttgaaacaga gtccattgga gtgggagtta gggagtgtag 3720tggatgccaa atatgttttt cttcagtgct taagagaact gtttcctgaa gtccagcttt 3780gaacataaac aggggtgtgg gttgggggag gagcttagga caaacctctc tgatgaaggt 3840cagcaataga ctgaagtctt gactgcatgg aagaggaaaa acatcagaac tgtctgacaa 3900tggaggggac agtgagctac gcacaactgc cagcggaggt gaacttgcac ctgcccaggc 3960cggatgaaca tcagcctgca agaactagtt gtttgagttg atttgcagtg ctctcaatgg 4020gcaagtgcca cattttccct ggcagagatc tccaaaaatt taaaacagaa taataatggc 4080tatatcgagt gttttctcag tattggagaa atgcttaggt cctatgatag cttcgggaca 4140tctttctgta attttcctca attaacgggt tggtaggggt aaatcttatg acacctttcc 4200accgtcgatt tgagatcagt tttaatggtt aaaatgttta ctctccttct gtcaaccctc 4260acctttttat ttacacccct cccttttttt ctgtacaggg agagaagaca tattgactct 4320gactggacac cctgattcct ccaaatatat ataccactgt gtattaatct ttctctcagt 4380gttttatagg agtactaaca tttattgctc tgtcaataat gaaaggctcg atgtaatata 4440gctgtaattt actttccata tgaatacagt ggctaggttc ataaaagaga attgtgtgag 4500tctgggatta ccacatctaa aacattattc tttaatggga taatacaatt cattgagcag 4560ctaccactta aaaaacttgc aggacagtta gagcctgcat ttctagttaa gatggatctt 4620gtaaatttaa aattggatta acattggagt gctggggtgg ctgcaataat ttgggggcta 4680actccatttg gtttccaaga tctcacttct gcattatctt tatggctctt taaaccagcc 4740acctagccaa tcaagggcaa ttcccatctc atccatcact caggtctttg taaagggtgc 4800agccaagctc tgcagacttt tgcaggattg tctagcctga gtaccgggct acttcttaaa 4860tgccgtcact cctgctgaga taaatgcgtc tttaaaaata gtctctgtgg caggtcactg 4920ggggacaatg tacagcattc tggccatcca cttctttttc acttcatgtt ctaccccaag 4980agactcccga tgtcggctgt ggagggttaa agggatgagg ctttcctttg tttagcaaat 5040ctgttcacag ttcttgatga tgtattttat gatgcccagc ttggaaatag ttgctttcca 5100tagtctcaac tgtattgtgt catctcctga tgctgatttt tgatcttttg ttttattaaa 5160aataattagt gaaagaggtg tgcctatctg tgaagtttgt agtacatcat cctgaggtca 5220tgtaacaagt aaaccccaac ccagcgttcc ctcctacgtt gtgttagttc attaaaacta 5280aataataaaa ataactgtaa gaaaacctta a 53111442362DNAHomo sapiens 144cactcagggc aagggtgtcc gacggctgga gcgttctgtt ttgaacccaa agtggatgat 60gctgtcagag ctgaactact gaaaggaggc tgtgaaaatt tcccatcttc tcattggcca 120tcagttgaga taagatggaa gactcttaca aggataggac ttcactgatg aagggtgcca 180aggacattgc cagagaggtg aagaaacaaa cagtaaagaa ggtgaatcaa gctgtggacc 240gagcccagga tgaatacacc cagaggtcct acagtcggtt ccaagatgaa gaagatgatg 300atgactacta cccggctgga gaaacctata atggtgaggc caacgatgac gaaggctcaa 360gtgaagccac tgaggggcat gatgaagatg atgagatcta tgagggggag tatcagggca 420tccccagtat gaaccaagcg aaggacagca tcgtgtcagt ggggcagccc aagggcgatg 480agtacaagga ccgacgggag ctggaatcag aaaggagagc tgacgaggaa gagttagccc 540agcagtatga gctgataatc caagaatgcg gtcatggtcg ttttcagtgg gcccttttct 600tcgtcctggg catggctctt atggcagacg gtgtagaggt gtttgtcgtt ggcttcgtgt 660tacccagtgc tgagacagac ctctgcatcc caaattcagg atctggatgg ctaggcagca 720tagtgtacct cgggatgatg gtgggggcgt tcttctgggg aggactggca gacaaagtgg 780gaaggaaaca gtctcttctg atttgcatgt ctgtcaacgg attctttgcc ttcctttctt 840catttgtcca aggttatggc ttctttctct tctgtcgctt actttctgga ttcgggattg 900gaggagccat acccactgtg ttctcgtact ttgctgaagt cctggcccgg gaaaagcggg 960gcgaacactt gagctggctc tgcatgttct ggatgatcgg tggcatctac gcctctgcca 1020tggcctgggc catcatcccg cactacgggt ggagcttcag catgggatcg gcctaccagt 1080ttcacagttg gcgtgtgttt gtcatcgtct gtgcactccc ctgtgtctcc tccgtggtgg 1140ccctcacatt catgcctgaa agcccacgat tcttgttgga ggttggaaaa catgatgaag 1200cttggatgat tctgaagtta attcatgaca ccaacatgag agcccggggt cagcctgaga 1260aggtcttcac ggtaaacaaa ataaaaactc ctaaacaaat agatgagctg attgaaattg 1320agagtgacac aggaacatgg tataggaggt gttttgttcg gatccgcacc gagctgtacg 1380gaatttggtt gacttttatg agatgtttca actacccagt cagggataat acaataaagc 1440ttacaattgt ttggttcacc ctgtcctttg ggtactatgg attatccgtt tggttccctg 1500atgtcattaa acctctgcag tccgatgaat atgcattgct aaccagaaat gtggagagag 1560ataaatatgc aaatttcact attaacttta caatggaaaa tcagattcat actggaatgg 1620aatacgacaa tggcagattc ataggggtca agttcaaatc tgtaactttc aaagactctg 1680tttttaagtc ctgcaccttt gaggatgtaa cttcagtgaa cacctacttc aagaactgca 1740catttattga cactgttttt gacaacacag attttgagcc atataaattc attgacagtg 1800aatttaaaaa ctgctcgttt tttcacaaca agacgggatg tcagattacc tttgatgatg 1860actatagtgc ctactggatt tattttgtca actttctggg gacattggca gtattgccag 1920ggaacattgt gtctgctctg ctgatggaca gaattgggcg cttaacaatg ctaggtggct 1980ctatggtgct ttcggggatc agctgtttct tcctttggtt cggcaccagt gaatccatga 2040tgataggcat gctgtgtctg tacaatggat tgaccatctc agcctggaac tctcttgacg 2100tggtcactgt ggaactgtac cccacagacc ggagggcaac aggctttggc ttcttaaatg 2160cgctatgcaa ggcagcagcc gtcctgggaa acttaatatt tggctctctg gtcagcatca 2220ccaaatcaat ccccatcctg ctggcttcta ctgtgctcgt gtgtggagga ctcgttgggc 2280tgtgcctgcc tgacacacga acccaggttc tgatgtaatg ggaaaaaaag ccatccttcc 2340tgcgtttctt cctcctgccc tg 23621452053DNAHomo sapiens 145catctttgat gagggcagag ctcacgttgc attgaagacg aaacctcggg gaggtcaggc 60gctgtctttc cttccctccc tgctcggcgg ctccaccaca gttgcaacct gcagaggccc 120ggagaacaca accctcccga gaagcccagg tccagagcca aacccgtcac tgacccccca 180gcccaggcgc ccagccactc cccaccgcta ccatggccga agacgcagac atgcgcaatg 240agctggagga gatgcagcga agggctgacc agttggctga tgagtcgctg gaaagcaccc 300gtcgtatgct gcaactggtt gaagagagta aagatgctgg tatcaggact ttggttatgt 360tggatgaaca aggagaacaa ctcgatcgtg tcgaagaagg catgaaccat atcaaccaag 420acatgaagga ggctgagaaa aatttaaaag atttagggaa atgctgtggc cttttcatat 480gtccttgtaa caagcttaaa tcaagtgatg cttacaaaaa agcctggggc aataatcagg 540acggagtggt ggccagccag cctgctcgtg tagtggacga acgggagcag atggccatca 600gtggcggctt catccgcagg gtaacaaatg atgcccgaga aaatgaaatg gatgaaaacc 660tagagcaggt gagcggcatc atcgggaacc tccgtcacat ggccctggat atgggcaatg 720agatcgatac acagaatcgc cagatcgaca ggatcatgga gaaggctgat tccaacaaaa 780ccagaattga tgaggccaac caacgtgcaa caaagatgct gggaagtggt taagtgtgcc 840cacccgtgtt ctcctccaaa tgctgtcggg caagatagct ccttcatgct tttctcatgg 900tattatctag taggtctgca cacataacac acatcagtcc acccccattg tgaatgttgt 960cctgtgtcat ctgtcagctt cccaacaata ctttgtgtct tttgttctct cttggtctct 1020ttctttccaa aggttgtaca tagtggtcat ttggtggctc taactccttg atgtcttgag 1080tttcattttt cattttctct cctcggtggc atttgctgaa taacaacaat ttaggaatgc 1140tcaatgtgct gttgattctt tcaatccaca gtattgttct tgtaaaactg tgacattcca 1200cagagttact gccacggtcc tttgagtgtc aggctctgaa tctctcaaaa tgtgccgtct 1260ttggttcctc atggctgtta tctgtcttta tgatttcatg attagacaat gtggaattac 1320ataacaggca ttgcactaaa agtgatgtga tttatgcatt tatgcatgag aactaaatag 1380atttttagat tcctacttaa acaaaaactt tccatgacag tagcatactg atgagacaac 1440acacacacac acaaaacaac agcaacaaca acagaacaac aacaaagcat gctcagtatt 1500gagacactgt caagattaag ttataccagc aaaagtgcag tagtgtcact tttttcctgt 1560caatatatag agacttctaa atcataatca tcctttttta aaaaaaagaa ttttaaaaaa 1620gatggatttg acacactcac catttaatca tttccagcaa aatatatgtt tggctgaaat 1680tatgtcaaat ggatgtaata tagggtttgt ttgctgcttt tgatggctac gttttggaga 1740gagcaatctt gctgtgaaac agtgtggatg taaattttat aaggctgact cttactaacc 1800accatttccc ctgtggtttg ttatcagtac aattctttgt tgcttaatct agagctatgc 1860acaccaaatt gctgagatgt ttagtagctg ataaagaaac cttttaaaaa aataatataa 1920atgaatgaaa tataaactgt gagataaata tcattatagc atgtaatatt aaattcctcc 1980tgtctcctct gtcagtttgt gaagtgattg acattttgta gctagtttaa aattattaaa 2040aattatagac tcc 20531462053DNAHomo sapiens 146catctttgat gagggcagag ctcacgttgc attgaagacg aaacctcggg gaggtcaggc 60gctgtctttc cttccctccc tgctcggcgg ctccaccaca gttgcaacct gcagaggccc 120ggagaacaca accctcccga gaagcccagg tccagagcca aacccgtcac tgacccccca 180gcccaggcgc ccagccactc cccaccgcta ccatggccga agacgcagac atgcgcaatg 240agctggagga gatgcagcga agggctgacc agttggctga tgagtcgctg gaaagcaccc 300gtcgtatgct gcaactggtt gaagagagta aagatgctgg tatcaggact ttggttatgt 360tggatgaaca aggagaacaa ctggaacgca ttgaggaagg gatggaccaa atcaataagg 420acatgaaaga agcagaaaag aatttgacgg acctaggaaa attctgcggg ctttgtgtgt 480gtccctgtaa caagcttaaa tcaagtgatg cttacaaaaa agcctggggc aataatcagg 540acggagtggt ggccagccag cctgctcgtg tagtggacga acgggagcag atggccatca 600gtggcggctt catccgcagg gtaacaaatg atgcccgaga aaatgaaatg gatgaaaacc 660tagagcaggt gagcggcatc atcgggaacc tccgtcacat ggccctggat atgggcaatg 720agatcgatac acagaatcgc cagatcgaca ggatcatgga gaaggctgat tccaacaaaa 780ccagaattga tgaggccaac caacgtgcaa caaagatgct gggaagtggt taagtgtgcc 840cacccgtgtt ctcctccaaa tgctgtcggg caagatagct ccttcatgct tttctcatgg 900tattatctag taggtctgca cacataacac acatcagtcc acccccattg tgaatgttgt 960cctgtgtcat ctgtcagctt cccaacaata ctttgtgtct tttgttctct cttggtctct 1020ttctttccaa aggttgtaca tagtggtcat ttggtggctc taactccttg atgtcttgag 1080tttcattttt cattttctct cctcggtggc atttgctgaa taacaacaat ttaggaatgc 1140tcaatgtgct gttgattctt tcaatccaca gtattgttct tgtaaaactg tgacattcca 1200cagagttact gccacggtcc tttgagtgtc aggctctgaa tctctcaaaa tgtgccgtct 1260ttggttcctc atggctgtta tctgtcttta tgatttcatg attagacaat gtggaattac 1320ataacaggca ttgcactaaa agtgatgtga tttatgcatt tatgcatgag aactaaatag 1380atttttagat tcctacttaa acaaaaactt tccatgacag tagcatactg atgagacaac 1440acacacacac acaaaacaac agcaacaaca acagaacaac aacaaagcat gctcagtatt 1500gagacactgt caagattaag ttataccagc aaaagtgcag tagtgtcact tttttcctgt 1560caatatatag agacttctaa atcataatca tcctttttta aaaaaaagaa ttttaaaaaa 1620gatggatttg acacactcac catttaatca tttccagcaa aatatatgtt tggctgaaat 1680tatgtcaaat ggatgtaata tagggtttgt ttgctgcttt tgatggctac gttttggaga 1740gagcaatctt gctgtgaaac agtgtggatg taaattttat aaggctgact cttactaacc 1800accatttccc ctgtggtttg ttatcagtac aattctttgt tgcttaatct agagctatgc 1860acaccaaatt gctgagatgt ttagtagctg ataaagaaac cttttaaaaa aataatataa 1920atgaatgaaa tataaactgt gagataaata tcattatagc atgtaatatt aaattcctcc 1980tgtctcctct gtcagtttgt gaagtgattg acattttgta gctagtttaa aattattaaa 2040aattatagac tcc 20531475PRTArtificial SequenceSNAP-25 antigen having a free carboxyl-terminus at the P1 residue of the scissile bond of the BoNT/A cleavage site 147Asp Glu Ala Asn Gln1 51486PRTArtificial SequenceSNAP-25 antigen having a free carboxyl-terminus at the P1 residue of the scissile bond of the BoNT/A cleavage site 148Ile Asp Glu Ala Asn Gln1 5

Claims

1. (canceled)

2. (canceled)

3. A method of quantifying picomolar amounts of botulinum neurotoxin serotype A (BoNT/A) activity in a sample, the method comprising: a. obtaining a sample comprising BoNT/A or suspected of comprising BoNT/A; b. contacting the sample with a plurality of cells from an established cell line susceptible to intoxication by about 50 pM or less of a BoNT/A and expressing a SNAP-25 comprising a BoNT/A cleavage site; c. measuring a reporter signal that corresponds to the amount of a SNAP-25 cleavage product in the plurality of cells; and d. determining the BoNT/A activity in the sample by comparing the reporter signal measured in step (c) relative to a dose-response curve generated by the method with a sample of known potency defined by mouse LD.sub.50 assay; wherein the method has an LOQ of <5 pM BoNT/A.

4. The method of claim 3, wherein a fluorophore is utilized to generate the reporter signal in step (c).

5. The method of claim 3, wherein a labeled antibody is utilized to generate the reporter signal in step (c).

6. The method of claim 5, wherein the labeled antibody is an isolated .alpha.-SNAP-25 antibody, comprising a heavy chain variable region comprising complementary determining regions (CDRs) comprising the amino acid sequences of at least one of SEQ ID NOs: 93, 96, and 100 and a light chain variable region comprising CDRs comprising the amino acid sequences of at least one of SEQ ID NOs: 105, 110 and 115.

7. The method of claim 3, wherein the method does not utilize fluorescence resonance energy transfer (FRET).

8. The method of claim 3, wherein the SNAP-25 is a non-naturally occurring SNAP-25.

9. The method of claim 8, wherein the established cell line has been engineered to express the non-naturally occurring SNAP-25.

10. The method of claim 9, wherein the non-naturally occurring SNAP-25 comprises at least one fluorophore.

11. The method of claim 9, wherein the non-naturally occurring SNAP-25 is green fluorescent protein.

12. The method of claim 3, wherein the sample is bulk BoNT/A drug substance.

13. The method of claim 3, wherein the sample is a formulated BoNT/A pharmaceutical product.

14. The method of claim 3, wherein the established cell line is a neuronal cell line.

15. The method of claim 3, wherein the established cell line is selected from the group comprising: SiMa and Neuro-2a cell lines.

16. The method of claim 15, wherein the established cell line is a Neuro-2a cell line.

17. The method of claim 3, wherein the method has an LOQ of <1 pM BoNT/A.

18. The method of claim 3, wherein the cell is susceptible to BoNT/A intoxication by about 10 pM or less of a BoNT/A.

19. The method of claim 3, wherein the cell is susceptible to BoNT/A intoxication by about 1 pM or less of a BoNT/A.

20. The method of claim 3, wherein the sample comprises about 1 ng or less of a BoNT/A.

21. The method of claim 3, wherein the sample comprises about 10 pM or less of a BoNT/A.

22. An established cell line expressing SNAP-25 and susceptible to intoxication by about 50 pM or less of a BoNT/A.

23-31. (canceled)