AFFINITY LIGANDS AND METHODS RELATING THERETO

Abstract

Affinity ligands useful for mild elution affinity chromatography, including affinity ligands specific for immunoglobulins M, A, and E, are disclosed as are method of identifying and using such affinity ligands.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a divisional of U.S. patent application Ser. No. 15/166,761, filed May 27, 2016, which claims the benefit of priority of U.S. Provisional Application No. 62/167,387, filed May 28, 2015, the entire content of which is incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The invention relates to affinity ligands, such as antibody affinity ligands, and methods for using, identifying, and making affinity ligands.

REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS-WEB

[0003] The official copy of the sequence listing is submitted electronically via EFS-Web as an ASCII formatted sequence listing with a file named 1010888-105410US_ST25.txt, created on May 26, 2016, and having a size of 103,871 bytes and is filed concurrently with the specification. The sequence listing contained in this ASCII formatted document is part of the specification and is herein incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

[0004] Affinity chromatography is a method of separating biochemical mixtures based on highly specific interactions between an affinity ligand and its target, such as that between antibody and antigen. An affinity ligand that selectively interacts with the desired target is immobilized onto a solid support in order to create an affinity matrix that can be used in a column format. Affinity chromatography can be used in a number of applications, including nucleic acid purification, protein purification from cell free extracts or cell culture supernatants, and purification from blood.

BRIEF SUMMARY OF THE INVENTION

[0005] The present disclosure provides methods of generating affinity ligands, uses of such affinity ligands, and specific affinity ligands.

[0006] In one aspect, provided are affinity ligands that bind specifically to a target molecule, wherein the specific binding strength of the affinity ligand to the target molecule is reduced under buffer conditions including (i) a pH of about 4.0 to about 5.5 or (ii) about 1-2 M MgCl.sub.2.

[0007] In some instances, the buffer condition may have a pH of about 4.0 to about 5.0. In some instances, the buffer condition may have a pH of about 4.0. In some instances, the buffer condition may have a pH of about 5.0. In some instances, the buffer condition may include 2 M MgCl.sub.2. In some instances, the buffer condition may include 2 M MgCl.sub.2 and a relatively neutral pH.

[0008] In some instances, the target molecule may be an immunoglobulin. In some instances, the target molecule may be an immunoglobulin selected from the group consisting of an immunoglobulin M (IgM), an immunoglobulin A (IgA), or an immunoglobulin E (IgE).

[0009] In some instances, the affinity ligand may be an immunoglobulin. For example, in some instances the affinity ligand may be a recombinant Fab fragment or Fab fragment derivative. In some instances, the affinity ligand may be an anti-IgE antibody comprising heavy chain complementarity determining regions CDR1, CDR2, and CDR3 sequences selected from any of the sequences set forth in FIG. 1A. In some instances, the affinity ligand may be an anti-IgE antibody having a light chain complementarity determining regions CDR1, CDR2, and CDR3 sequences selected from any of the sequences set forth in FIG. 1B. In some instances, the affinity ligand may be an anti-IgA antibody comprising heavy chain complementarity determining regions CDR1, CDR2, and CDR3 sequences selected from any of the sequences set forth in FIG. 2A. In some instances, the affinity ligand may be an anti-IgA antibody comprising light chain complementarity determining regions CDR1, CDR2, and CDR3 sequences selected from any of the sequences set forth in FIG. 2B. In some instances, the affinity ligand may be an anti-IgM antibody comprising heavy chain complementarity determining regions CDR1, CDR2, and CDR3 sequences selected from any of the sequences set forth in FIG. 3A. In some instances, the affinity ligand may be an anti-IgM antibody comprising light chain complementarity determining regions CDR1, CDR2, and CDR3 sequences selected from any of the sequences set forth in FIG. 3B.

[0010] In some instances, the affinity ligand may be linked to a solid support. In some instances, the solid support may be a bead or a sample plate. In some instances, the bead may be an agarose bead, a polystyrene bead, a polymethacrylate bead, a polyacrylamide bead, a magnetic bead, or a paramagnetic bead.

[0011] In another aspect, provided are methods of isolating a target molecule, the method comprising the steps of: providing a solid support linked to an affinity ligand; contacting the solid support with a sample containing the target molecule; washing the solid support with a wash buffer to remove unbound components of the sample; and eluting bound target molecule from the solid support with an elution buffer comprising (i) a pH of about 4.0 to about 5.5 or (ii) about 1-2 M MgCl.sub.2.

[0012] In some instances, the elution buffer may include a pH of about 4.0 to about 5.5 and a relatively low salt concentration. In some instances, the elution buffer may include about 1 M to 2 M MgCl.sub.2 and a relatively neutral pH. In some instances, the elution buffer may include about 1 M to 2 M MgCl.sub.2 and a pH of about 6.0 to 8.0. In some instances, the eluting may be a single-step elution with an elution buffer comprising (i) a pH of about 4.0 to about 5.5 or (ii) about 1-2 M MgCl.sub.2. In some instances, the eluting may be a multiple-step elution with a plurality of elution buffers comprising (i) a pH of about 4.0 to about 5.5 or (ii) about 1-2 M MgCl.sub.2, wherein the plurality of elution buffers are applied to the solid support sequentially, wherein elution buffers having higher salt concentrations are applied after elution buffers having lower salt concentrations and elution buffers having lower pH are applied after elution buffers having higher pH. In some instances, the eluting may be a gradient elution with an elution buffer having a gradient of linearly increasing salt concentration during the time of the eluting, wherein the maximum salt concentration is about 1-2 M MgCl.sub.2. In some instances, the eluting may be a gradient elution with an elution buffer having a gradient of linearly decreasing pH during the time of the eluting, wherein the minimum pH is about 4.0. In some instances, the wash buffer may have a pH of 6.0-8.0. In some instances, the wash buffer may have a relatively low salt concentration.

[0013] In some instances, the affinity ligand may be an immunoglobulin. In some instances, the target molecule may be an immunoglobulin M (IgM), an immunoglobulin A (IgA), or an immunoglobulin E (IgE). In some instances, the affinity ligand may be an anti-IgE antibody comprising heavy chain complementarity determining regions CDR1, CDR2, and CDR3 sequences selected from any of the sequences set forth in FIG. 1A. In some instances, the affinity ligand may be an anti-IgE antibody comprising light chain complementarity determining regions CDR1, CDR2, and CDR3 sequences selected from any of the sequences set forth in FIG. 1B. In some instances, the affinity ligand may be an anti-IgA antibody comprising heavy chain complementarity determining regions CDR1, CDR2, and CDR3 sequences selected from any of the sequences set forth in FIG. 2A. In some instances, the affinity ligand may be an anti-IgA antibody comprising light chain complementarity determining regions CDR1, CDR2, and CDR3 sequences selected from any of the sequences set forth in FIG. 2B. In some instances, the affinity ligand may be an anti-IgM antibody comprising heavy chain complementarity determining regions CDR1, CDR2, and CDR3 sequences selected from any of the sequences set forth in FIG. 3A. In some instances, the affinity ligand may be an anti-IgM antibody comprising light chain complementarity determining regions CDR1, CDR2, and CDR3 sequences selected from any of the sequences set forth in FIG. 3B.

[0014] In another aspect, provided are methods of selecting an affinity ligand that specifically binds to a target molecule under neutral buffer conditions and has reduced binding strength to the target molecular under mild elution conditions, the method including the steps of: expressing a naive affinity ligand library to produce a plurality of affinity ligands; providing a solid support linked to a target; contacting the solid support with the plurality of affinity ligands; washing the solid support with a wash buffer to remove unbound affinity ligands, wherein the wash buffer comprises neutral buffer conditions; contacting the solid support with an elution buffer comprising (i) a pH of about 4.0 to about 5.5 or (ii) about 1-2 M MgCl.sub.2; and identifying affinity ligands that substantially dissociate from the solid support in the elution buffer.

[0015] In some instances, the affinity ligand library may not be preselected for characteristics favoring reduced binding strength to the target molecule under mild elution conditions. In some instances, the plurality of affinity ligands may be encoded by a plurality of nucleic acid sequences. In some instances, the plurality of nucleic acid sequences includes a heterologous promoter operably linked thereto. In some instances, the plurality of affinity ligands may be expressed on a plurality of phage.

[0016] In some instances, the elution buffer may have a pH of about 4.0 to about 5.5 and a relatively low salt concentration. In some instances, the elution buffer may include about 1 M to 2 M MgCl.sub.2 and may have a relatively neutral pH. In some instances, the elution buffer may include about 1 M to 2 M MgCl.sub.2 and may have a pH of about 6.0 to 8.0. In some instances, the wash buffer may have a pH of 6.0-8.0. In some instances, the wash buffer may have a relatively low salt concentration.

[0017] In some instances, the target may be an immunoglobulin. In some instances, the target may be an immunoglobulin M (IgM), an immunoglobulin A (IgA), or an immunoglobulin E (IgE).

[0018] In some instances, the plurality of affinity ligands may be a plurality of antibodies or derivatives thereof. In some instances, the plurality of affinity ligands is a plurality of Fab fragments or derivatives thereof. In some instances, the affinity ligand identified may be encoded by a polynucleotide comprising a nucleic acid sequence encoding heavy chain complementarity determining regions CDR1, CDR2, and CDR3 sequences selected from any of the sequences set forth in FIG. 1A. In some instances, the affinity ligand identified may be encoded by a polynucleotide comprising a nucleic acid sequence encoding light chain complementarity determining regions CDR1, CDR2, and CDR3 sequences selected from any of the sequences set forth in FIG. 1B. In some instances, the affinity ligand identified may be encoded by a polynucleotide comprising a nucleic acid sequence encoding heavy chain complementarity determining regions CDR1, CDR2, and CDR3 sequences selected from any of the sequences set forth in FIG. 2A. In some instances, the affinity ligand identified may be encoded by a polynucleotide comprising a nucleic acid sequence encoding light chain complementarity determining regions CDR1, CDR2, and CDR3 sequences selected from any of the sequences set forth in FIG. 2B. In some instances, the affinity ligand identified may be encoded by a polynucleotide comprising a nucleic acid sequence encoding heavy chain complementarity determining regions CDR1, CDR2, and CDR3 sequences selected from any of the sequences set forth in FIG. 3A. In some instances, the affinity ligand identified may be encoded by a polynucleotide comprising a nucleic acid sequence encoding light chain complementarity determining regions CDR1, CDR2, and CDR3 sequences selected from any of the sequences set forth in FIG. 3B.

[0019] In another aspect, provided are kits including the affinity ligand described above.

[0020] It will be appreciated from a review of the remainder of this application that further methods and compositions are also part of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] FIG. 1A and FIG. 1B show heavy and light chain CDR sequences, respectively, for anti-IgE antibodies according to some examples.

[0022] FIG. 2A and FIG. 2B show heavy and light chain CDR sequences, respectively, for anti-IgA antibodies according to some examples.

[0023] FIG. 3A and FIG. 3B show heavy and light chain CDR sequences, respectively, for anti-IgM antibodies according to some examples.

[0024] FIGS. 4A-4I show heavy and light chain sequences for anti-IgM antibodies according to some examples.

[0025] FIG. 5 shows ELISA results for a first set of anti-IgM antibody affinity ligands assessing binding to and elution of target molecules according to one example.

[0026] FIG. 6 shows ELISA results for a second set of anti-IgM antibody affinity ligands assessing binding to and elution of target molecules according to one example.

[0027] FIG. 7 shows ELISA results for a first set of anti-IgA antibody affinity ligands assessing binding to and elution of target molecules according to one example.

[0028] FIG. 8 shows ELISA results for a second set of anti-IgA antibody affinity ligands assessing binding to and elution of target molecules according to one example. Controls N1-CD33-6.times.His and BSA are also shown.

[0029] FIG. 9 shows ELISA results for a set of anti-IgE antibody affinity ligands assessing binding specificity for target molecules according to one example.

[0030] FIG. 10 shows elution profiles of purified AbD18705 hIgM target molecule from affinity ligand columns according to one example. The affinity column on the left uses anti-IgM antibody ligand AbD20775.2, and the affinity column on the right uses anti-IgM antibody ligand AbD20771.2. Collected fractions (A#) are shown across the bottom of each graph.

[0031] FIG. 11 shows SDS-PAGE analysis of purified AbD18705 hIgM target molecule elution fractions according to one example. The left gel shows the purified AbD18705 hIgM from the indicated fractions (each fraction shown under reducing and under oxidizing conditions). Purified human IgM (product number OBT1524) is shown as a control in the gel on the right, again under reducing and oxidizing conditions.

[0032] FIG. 12 shows an overlay of size exclusion chromatography runs for purified AbD18705 hIgM target molecule elution fractions identified in FIG. 10 according to one example.

[0033] FIG. 13 shows a graph illustrating the results of an ELISA assay assessing the activity and specificity of the purified AbD18705 hIgM target molecule ("His-GFP) for its antigen GFP according to one example. Controls GST, N1-CD33-6.times.His, and BSA are also shown.

DEFINITIONS

[0034] "Affinity ligand" or "ligand" refers to a composition (such as, for example, an antibody or non-antibody protein), that binds specifically to a specific substance, such as a protein, protein complex, or organic compound having a defined structure.

[0035] The term "solid support" is used herein to denote a solid inert surface or body to which an agent, such as an antibody or an antigen, that is reactive in any of the binding reactions described herein can be immobilized. The term "immobilized" as used herein denotes a molecularly-based coupling that is not dislodged or de-coupled under any of the conditions imposed during any of the steps of the assays described herein. Such immobilization can be achieved through a covalent bond, an ionic bond, an affinity-type bond, or any other covalent or non-covalent bond.

[0036] The term "antibody" or "immunoglobulin" refers to an immunoglobulin, composite, or fragmentary form thereof. The term may include but is not limited to polyclonal or monoclonal antibodies of the classes IgA, IgD, IgE, IgG, and IgM, derived from human or other cell lines, including natural or genetically modified forms such as humanized, human, single-chain, chimeric, synthetic, recombinant, hybrid, mutated, grafted, and in vitro generated antibodies and fragments thereof. "Antibody" may also include composite forms including but not limited to fusion proteins containing an immunoglobulin moiety. "Antibody" may also include non-quaternary antibody structures (such as camelids and camelid derivatives). "Antibody" may also include antibody fragments such as Fab (fragment-antigen binding), F(ab')2, Fv, scFv, Fd, dAb, Fc and other compositions that retain antigen-binding function. In addition, the term "antibody" includes aggregates, polymers, and conjugates of immunoglobulins or their fragments, where the molecules largely retain binding affinity for their epitope(s). Further, an "antibody" may be modified, such as, for example, by linking to a chemical or peptide moiety or detectable tag moiety.

[0037] The term "complementarity determining region" or "CDR" (also known as "hypervariable region" or "HVR") refers to an immunoglobulin hypervariable domain that determines specific binding of an immunoglobulin to an epitope. The variable regions of both the heavy and light chains of an antibody each generally contain three CDRs. Antibodies with different specificities have different CDRs, while antibodies of the exact same specificity may have identical CDRs.

[0038] A "constant region" refers to a region in the heavy and light chains of an antibody having relatively less variability compared to the N' terminal variable region of the heavy and light chains of an antibody. On the heavy chains, the constant region is generally the same in all antibodies of the same isotype and differs in antibodies of different isotypes. There are two primary types of light chains (kappa and lambda), each with a distinct constant region.

[0039] "Neutral buffer" or "neutral buffer condition" refers to a buffer having approximately physiological pH. Such buffers/conditions allow binding of proteins to an affinity column without resulting in substantial protein denaturation or aggregation. A neutral buffer or neutral buffer condition may permit near optimal interaction between an affinity ligand and a target molecule. A neutral buffer condition or neutral buffer generally has a pH in the range of about 6.0 to 8.0.

[0040] "Mild elution condition" or "mild elution buffer" refers to a buffer in which an affinity ligand that specifically binds to a target molecule dissociates from the target molecule (such as on an affinity matrix) without resulting in substantial protein denaturation or aggregation of the target molecule. This is in contrast to the harsh conditions that are typically applied in elution of a target molecule, such as low pH (about pH 2 to 3.5, for example). Such mild elution buffers may include relatively high pH (pH.gtoreq.4.0) or relatively high salt or ionic strength.

[0041] "Physiological pH" refers to the pH of human blood, which is about 7.4. A pH in the range of pH 7.0 to pH 7.8 may be considered approximately physiological pH.

[0042] "Naive expression library," for the purposes of expressing affinity ligands, refers to an expression library that expresses a large number of recombinant proteins, polypeptides, or peptides that have a large diversity of structure or specificity in their binding domains. A naive library is one generated from synthetic or natural ligand repertoires that have not previously subjected to selections for increased affinity for a target. A naive expression library is generated from a plurality of nucleic acid sequences that encode a plurality of affinity ligands or at least a plurality of target binding regions.

[0043] "Relatively neutral pH" refers to a pH of about pH 6.0 to 8.0.

[0044] A "solid support" refers to a material or group of materials having a rigid or semi-rigid surface or surfaces. In some embodiments, the solid support takes the form of thin films or membranes, beads, bottles, dishes, fibers, woven fibers, shaped polymers, particles, and microparticles, including but not limited to, microspheres. A solid support can be formed, for example, from an inert solid support of natural material, such as glass and collagen, or synthetic material, such as acrylamide, cellulose, nitrocellulose, silicone rubber, polystyrene, polyethylene vinyl acetate, polypropylene, polymethacrylate, polyethylene, polysilicates, polyethylene oxide, polycarbonates, teflon, fluorocarbons, nylon, polyanhydrides, polyglycolic acid, polylactic acid, polyorthoesters, polypropylfumarate, glycosaminoglycans, and polyamino acids. In some cases, some functional groups naturally present on the surface of a carrier (for example, carboxylic acid (--COOH), free amine (--NH2), and sulfhydryl (--SH) groups) can be used for peptide linkage. In case no such functional group is naturally available, a desired functional group, such as a carboxylic acid group, or a moiety known to be a partner of a binding interaction (such as avidin that is capable of binding biotin) may be attached to such solid support. In some embodiments, the solid support is a carboxylated latex or magnetic microsphere.

[0045] The phrase "specific binding" or "binds specifically" refers to a binding reaction where two members of a binding pair (for example, an antibody and a molecule comprising the antibody's target epitope) bind to each other with an affinity that is at least 10-fold better than the members' affinity for other components of a heterogeneous mixture (for example, a hybridoma culture supernatant or other mixture of proteins).

[0046] The term "variable region" refers to an N' terminal region of each of the heavy and light chains of an antibody that has relatively more variability compared to the constant region(s) of the heavy and light chains of an antibody. The variable region contains the CDRs.

DETAILED DESCRIPTION OF THE INVENTION

[0047] It has been discovered that affinity ligands (such as antibodies) can be identified for use in immunoaffinity purification of target molecules (such as immunoglobulins M, A, or E) such that mild conditions can be used to elute the target molecule to which the affinity ligand specifically binds, thereby avoiding harsh elution conditions used previously. Certain examples and features of the present disclosure relate to methods to identify affinity ligands, such as antibodies, that are especially useful for mild elution immunoaffinity chromatography. Such ligands, when attached to a solid support, allow one-step purification of target molecules using mild elution conditions. The use of mild elution conditions circumvents the typically harsh elution conditions used in this type of chromatography that may lead to denaturing or aggregation of target molecules (such as low pH, for example, pH 3.0). The described methods utilize in vitro enrichment methods to permit selection of affinity ligands for which mild elution conditions can be used. The selection of such ligands is performed by a first binding step under conditions that allow binding in standard/neutral buffer conditions (such as neutral pH and/or low salt), followed by a subsequent elution step that is performed using mild elution conditions. The described methods permit specific selection and enrichment and finally isolation of affinity ligands having the desired characteristics. As such, affinity ligands of interest can be generated more rapidly and efficiently using the described methods.

[0048] Certain examples and features of the present disclosure relate to affinity ligands, such as antibodies, that exhibit the property of specifically binding to a target under neutral conditions and releasing the target under mild elution conditions. These affinity ligands can be selected for using the method described above. Examples of affinity ligands that are specific for human immunoglobulin M (IgM), human immunoglobulin A (IgA), and human immunoglobulin E (IgE) are described.

[0049] Certain examples and features of the present disclosure relate to methods of using such affinity ligands to purify targets using mild elution conditions. Affinity ligands may be used linked to a solid support as an affinity purification column. For example, some targets that are sensitive to harsh elution conditions (for example, pH 3.0) may be successfully purified using an affinity column generated using the affinity ligands that allow elution of targets under mild elution conditions. For example, such affinity ligands are useful for isolating IgM, IgA, or IgE molecules as these molecules may be sensitive to denaturation when affinity purified using harsh elution conditions. Other molecules that are sensitive to denaturation can be purified accordingly, once an affinity ligand has been isolated using the methods described in this disclosure.

[0050] I. Affinity Ligands

[0051] Affinity ligand compositions are described herein that bind specifically to a target and from which target can be eluted using mild elution conditions. As described further below, the affinity ligand may be an antibody, antibody-like molecule, or other affinity-binding molecule. For ease of description, this disclosure will sometimes describe examples and features of the affinity ligands in the context of antibody affinity ligands. However, this disclosure is not limited to affinity ligands that are antibodies.

[0052] The affinity ligand may be selected from a variety of different types of protein or non-protein compositions. In certain cases, an affinity ligand is an antibody, antibody-like molecule, or any other affinity-binding molecule derived from a naive expression library as described below in Section II. For example, the antibody may be a monoclonal antibody, a Fab fragment, a F(ab')2, an Fv, a scFv, an Fd, a dAb, an Fc fragment, a VHH, or other fragments thereof that retain antigen-binding function. In some examples, affinity ligand may be recombinant antibodies having heterologous constant and variable domains; for example, generated by a recombinant protein expression library. In some instances, the affinity ligand may be a recombinant Fab fragment. For example, the Fab fragment may have the variable domain and the first constant domain (Fd chain) of one heavy chain plus one complete light chain (L chain). The Fd and L chain are linked by strong non-covalent interactions and can be covalently linked by a disulfide bond. The polynucleotide sequence encoding the recombinant Fab fragment may be subcloned into an expression vector containing a heterologous promoter to drive expression of the recombinant Fab fragment. The Fab fragment may monovalent, bivalent, or multivalent. In some instances, the Fab fragment is monovalent. In some examples, the affinity ligand is a single-chain variable (scFv) fragment or a bivalent scFv fragment (diabody). ScFv fragments typically have one VH and one VL chain expressed as a single polypeptide joined by a peptide linker. The polypeptide linker stabilizes the interaction between the VH and VL chains. The polynucleotide sequence encoding the recombinant scFv fragment may be subcloned into an expression vector containing a heterologous promoter to drive expression of the recombinant scFv fragment. In some instances, the affinity ligand may be a recombinant antibody or protein that specifically binds to targets in a manner similar to antibodies, or fragment thereof that retains antigen-binding function. The affinity ligand may be a recombinant protein that contains at least three complementarity determining regions (CDRs) that cause the specific binding of the affinity ligand to the target; for example, three heavy chain CDRs and, in some examples, contains six CDRs (three heavy chain and three light chain). In some instances, the antibody may be a variable domain of heavy chain (VHH) antibody or a nanobody (a monomeric variable domain antibody). The VHH or nanobody may be encoded by a singly polypeptide.

[0053] In some examples, the affinity ligand is a camelid antibody or camelid nanobody. Camelid antibodies are certain IgG antibodies from the mammalian family of camel and dromedary (Camelus bactrianus and Camelus dromaderius) family, including new world members such as llama species (such as Lama paccos, Lama glama and Lama vicugna), that lack light chains. See, for example, International Appl. WO 94/04678. The small single variable domain (VHH) of the camelid antibody can be used to as the basis of a low molecular weight antibody-derived protein known as a "camelid nanobody" having high affinity for a target. See U.S. Pat. No. 5,759,808; see also Stijlemans, B. et al., 2004 J Biol Chem 279: 1256-1261; Dumoulin, M. et al., 2003 Nature 424: 783-788; Pleschberger, M. et al. 2003 Bioconjugate Chem 14: 440-448; Cortez-Retamozo, V. et al. 2002 Int J Cancer 89: 456-62; and Lauwereys, M. et al. 1998 EMBO J 17: 3512-3520. Engineered libraries of camelid antibodies and antibody fragments are commercially available.

[0054] In some cases, the affinity ligand can be a compound or non-antibody protein that specifically binds to targets in a manner similar to antibodies. Certain of these "antibody mimics" use non-immunoglobulin protein scaffolds as alternative protein frameworks for the variable regions of antibodies. For example, the affinity ligand can be a monobody, which are small antibody mimics using the scaffold of a fibronectin type III domain (FN3). FN3 scaffold functions as an effective framework onto which loops for specific building functions can be grafted. For example, the affinity ligand may utilize the tenth FN3 unit of human fibronectin as scaffold. It is small, monomeric, and does not have disulfide bonds. FN3-based antigen-binding molecules can be prepared using methods described in the art. For example, see Koide et al., J. Mol. Biol. 284: 1141-1151, 1998; Koide et al., Proc. Natl Acad. Sci. USA 99:1253-1258, 2002; and Batori et al., Protein Eng. 15:1015-20, 2002, and U.S. Pat. Nos. 6,818,418 and 7,115,396. In another example, the affinity ligand may be a single polypeptide chain binding molecule that contains the antigen binding sites of both the heavy and light variable regions of an antibody connected by a peptide linker and will fold into a structure similar to that of the two peptide antibody. See, for example, U.S. Pat. No. 5,260,203. Also, the affinity ligand may be a recombinant protein containing derivative sequences of one or more loops of cytochrome b562 that are selected for binding specificity to the target. See, for example, Ku et al., Proc. Natl. Acad. Sci. U.S.A. 92(14):6552-6556 (1995). In another example, the affinity ligand may be an antibody mimic based on a lipocalin scaffold, in which one or more of the hypervariable loops of the lipocalin protein are randomized and selected for specific binding to the target. See, for example, Beste et al., Proc. Natl. Acad. Sci. U.S.A. 96(5):1898-1903 (1999). An example of such antibody mimetics are Anticalins.RTM., which are small, single chain peptides, typically between 160 and 180 residues. In addition, the affinity ligand may be a synthetic antibody mimic using the rigid, non-peptide organic scaffold of calixarene to which are attached multiple variable peptide loops used as binding sites. See, for example, U.S. Pat. No. 5,770,380. In some examples, the affinity ligand may be an antibody-like binding peptidomimetic. See, for example, Murali et al., Cell. Mol. Biol. 49(2):209-216 (2003). Also, in some examples, the affinity ligand may include a scaffold derived from one or more A-domains. For example, the affinity ligand may include multiple A-domains, each of which binding independently to a distinct epitope of the target. Such affinity ligands can be generated using methods described in, for example, Gliemann et al., Biol. Chem. 379:951-964, 1998; Koduri et al., Biochemistry 40:12801-12807, 2001 and Silverman et al., Nat Biotechnol. 23:1556-61, 2005. Other exemplary non-antibody scaffolds for use as ligands include darpins, affimers, cystine-knot mini-proteins, affilins, and peptides or non-protein-based scaffolds like aptamers.

[0055] In some instances, the affinity ligand may comprise an affinity tag or moiety. The affinity tag or moiety may be useful for purification of the affinity ligand or attachment to a solid support. For example, the affinity ligand may include at least one of a FLAG.RTM. peptide, 6.times.-Histidine (6.times.His) peptide, etc. In some cases, the affinity ligand is chemically modified to facilitate attachment of the ligand to a solid support.

[0056] In one aspect, the affinity ligand binds specifically to its target under neutral buffer conditions. Neutral buffer conditions may include a pH of approximately physiological pH, or relatively neutral pH, such as, for example a pH of about 6.0, 6.2, 6.5, 6.8, 7.1, 7.3, 7.5, 7.8, 7.9, 8.1, or a pH of about 6.0 to about 8.0. In some instances, the neutral buffer condition also comprises a relatively low salt concentration and/or ionic strength. For example, the salt concentration may be approximately physiological salt concentration. In some cases, the neutral buffer conditions include approximately physiological ionic strength. In some instances, neutral buffer conditions may be used for a wash buffer. In such instances, the wash buffer may have somewhat higher salt concentration or ionic strength to improve stringency and reduce non-specific binding of non-targets to the affinity ligand. In some instances, the neutral buffer condition does not include a salt. In certain instances, the neutral buffer condition may include detergent to reduce non-specific binding of non-targets to the affinity ligand. In some instances, the neutral buffer condition comprises a buffering agent. In one example, a neutral wash buffer may be phosphate buffered saline (PBS). In another example, the neutral wash buffer may be PBS containing about 0.05-1.0% Tween-20.RTM. (for example, 0.1%). Other solutions and detergents are contemplated.

[0057] In another aspect, under mild elution conditions, the specific binding of the affinity ligand to its target is reduced such that the affinity ligand and the target substantially dissociate from each other. In one aspect, the mild elution conditions may comprise a pH of about 4.0 to about 5.5. In another aspect, the mild elution conditions may comprise about 1-2 M MgCl.sub.2.

[0058] In one aspect, mild elution conditions may comprise a pH in the range of about 4.0 to about 5.5. For example, mild elution conditions may include a pH of at least about 4.0 but less than or equal to about 5.5. The mild elution condition may include a pH greater than or equal to about 4.0, a pH of about 4.5, a pH of about 5.0, or a pH of about 5.5, or any pH within the range of about 4.0 to about 5.5. Where the pH is in the range of about 4.0 to 5.5, the elution buffer may further comprise a relatively low salt concentration. In some instances, the elution buffer may contain salt conditions relatively similar to physiological salt conditions. In some instances, the salt concentration may be 25 mM, 50 mM, 100 mM, 125 mM, 150 mM, 175 mM, 200 mM, 225 mM, or 250 mM. In one example, the elution buffer may include about 150 mM NaCl. In some cases, the mild elution conditions include approximately physiological ionic strength. In some instances the elution buffer may further include a buffering agent. Exemplary buffering agents include citrate, sodium acetate, and sodium phosphate buffered saline (PBS). In one example, the elution buffer may contain 100 mM citrate buffer. In another example, the elution buffer may contain 100 mM sodium acetate. In another example, the elution buffer may contain 1.times.PBS. In one example, the elution buffer may contain 150 mM NaCl and 100 mM citrate buffer.

[0059] In another aspect, mild elution conditions may comprise a salt concentration of about 1 M to 2 M MgCl.sub.2. For example, the salt concentration may be about 0.8 M, about 1 M, about 1.2 M, about 1.4 M, about 1.6 M, about 1.8 M, about 2 M, or about 2.2 M. In one example, the mild elution condition comprises 2 M MgCl.sub.2. In some instances, the mild elution conditions may further comprise a relatively neutral pH, or an approximately physiological pH, or a pH of about 6.8 to about 7.9, or a pH of about 6.0 to about 8.0, or any pH with these ranges. For example, the pH may be about 6.0, about 6.2, about 6.4, about 6.6, about 6.8, about 7.0, about 7.2, about 7.4, about 7.6, about 7.8, or about 8.0, when the mild elution condition includes a salt concentration of about 1 M to 2 M MgCl.sub.2. In some instances, the elution buffer may further include a buffering agent. Exemplary buffering agents include citrate, sodium acetate, and phosphate buffered saline (PBS). The buffering agent may be of sufficient concentration to provide pH buffering. In one example, the elution buffer may contain 100 mM citrate. In another example, the elution buffer may contain 100 mM sodium acetate. In another example, the elution buffer may contain 1.times.PBS.

[0060] It is understood that, where the mild elution condition comprises a pH in the range of about 4.0 to 5.5, the neutral buffer conditions generally have a higher pH of approximately neutral pH or approximately physiological pH. It is also understood that, where the mild elution condition comprises about 1M to 2M MgCl.sub.2, the neutral buffer conditions generally have a lower salt concentration or ionic strength.

[0061] In one aspect, the affinity ligand binds specifically to a target epitope of a target molecule. The target epitope can be a portion of a target molecule, such as a protein, nucleic acid, or other biological molecule. For example, the target may be a protein or other molecule that is sensitive to harsh buffer conditions, including low pH (such as pH 3.0). In some instances, a target may denature, dissociate into individual subunits, or dissociate from cofactors under harsh buffer conditions. In some examples, the target molecule may be an immunoglobulin (antibody), such as an immunoglobulin G (IgG), an immunoglobulin M (IgM), an immunoglobulin A (IgA), or an immunoglobulin E (IgE). In some instances, the affinity ligand may be a Fab fragment that binds specifically to an IgG, an IgM, an IgA, or an IgE.

[0062] For example, the affinity ligand may be a Fab fragment that binds specifically to an IgE having any of the heavy and light chain CDR sequences as set forth in FIG. 1A and FIG. 1B, respectively. In some instances, the antibodies specific for IgE may contain a combination of CDR1, CDR2, and CDR3 sequences as set forth in FIG. 1A or FIG. 1B. In some examples, such affinity ligand antibodies will have reduced binding to IgE under mild elution conditions, such as at a pH equal to or greater than about 4.0 and less than or equal to about 5.5, such that the IgE elutes from the affinity agent. For example, as described in Example 2, certain anti-IgE antibodies substantially dissociate from their target under elution conditions comprising pH 4.0 and pH 5.0. In some instances, the elution buffer conditions also comprise relatively low salt, such as 150 mM NaCl, and a buffering agent, such as citrate. Other suitable salts and buffering agents are also contemplated.

[0063] In another example, the affinity ligand may be a Fab fragment that binds specifically to an IgA having any of the heavy and light chain CDR sequences as set forth in FIG. 2A and FIG. 2B, respectively. In some instances, the antibodies specific for IgA may contain a combination of CDR1, CDR2, and CDR3 sequences as set forth in FIG. 2A or FIG. 2B. In some examples, such affinity ligand antibodies will have reduced binding to IgA under mild elution conditions, such as at a pH equal to or greater than about 4.0 and less than or equal to about 5.5, such that the IgE elutes from the affinity agent. For example, as described in Example 2, certain anti-IgA antibodies substantially dissociate from their target under elution conditions comprising pH 4.0 and pH 5.0. In some instances, the elution buffer conditions also comprise relatively low salt, such as 150 mM NaCl, and a buffering agent, such as citrate. Other suitable salts and buffering agents are also contemplated.

[0064] In another example, the affinity ligand may be a Fab fragment that binds specifically to an IgM having any of the heavy and light chain CDR sequences as set forth in FIG. 3A and FIG. 3B, respectively. In some examples, the affinity ligand may have at least one of the heavy chain sequences and at least one of the light chain sequences shown in FIGS. 4A-4I. In some instances, the affinity ligand may have at least one heavy and light chain sequences pair as shown in FIGS. 4A-4I. In some instances, the antibodies specific for IgM may contain a combination of CDR1, CDR2, and CDR3 sequences as set forth in FIG. 3A or FIG. 3B. In some examples, such affinity ligand antibodies will have reduced binding to IgM under mild elution conditions, such as at a pH equal to or greater than about 4.0 and less than or equal to about 5.5, such that the IgM elutes from the affinity agent. For example, as described in Example 2, certain anti-IgM antibodies substantially dissociate from their target under elution conditions comprising pH 4.0 and pH 5.0. In some instances, the elution buffer conditions also comprise relatively low salt, such as 150 mM NaCl, and a buffering agent, such as citrate. Other suitable salts and buffering agents are also contemplated.

[0065] In certain examples, the affinity ligand may have CDR sequences similar to those identified in this disclosure except varying in amino acid sequence at one or two amino acid positions. In some instances, the variance in sequence is a conservative amino acid change.

[0066] While this disclosure describes specific examples and features of the affinity ligands that are specific for immunoglobulin targets, affinity ligands specific for other types of ligands are also contemplated.

[0067] II. Selection Methods

[0068] The described selection methods allow for fast and efficient identification of affinity ligands, in particular antibodies, for use in affinity chromatography methods using mild elution conditions. In some embodiments, a plurality of potential affinity ligands are provided and processed such that ligands are selected that specifically bind to a target under neutral buffer conditions and display weak specific binding strength to the target under mild elution conditions.

[0069] A variety of methods are known and can be used for expressing a plurality of affinity ligands. In particular, naive expression libraries are useful for expressing a large number of recombinant proteins, polypeptides, and peptides, including, for example, antibodies, that have a large diversity of structure or specificity in their binding domains. A naive library is one generated from synthetic or natural ligand repertoires that have not previously subjected to selections for increased affinity for a target. In some instances, a naive library is generated from a plurality of nucleic acid sequences that encode a plurality of affinity ligands or at least a plurality of target binding regions. In some instances, the nucleic acid sequences encoding the target binding regions of the plurality of affinity ligands may have randomized sequences to generate binding regions in the plurality of affinity ligands that have randomized amino acid sequences. In some instances, the expression library expresses proteins or polypeptides having a structural domain with a common amino sequence and a binding domain having variable sequences such that the proteins or polypeptides expressed by the library may display a large number of distinct binding domains based on sequence variability. As such, different proteins or polypeptides expressed by the library may have different binding specificities for different targets under different conditions. In some examples, the expression libraries express hundreds of thousands, millions, or billions of different proteins or polypeptides, each of which may have a distinct binding affinity and, thus, may bind to different targets with different degrees of specificity under different conditions. Expression libraries useful for expressing a plurality of affinity ligands for use in this method include phage display libraries, yeast display libraries, ribosome display, mRNA display or other selection system, or any other recombinant expression library capable of expressing a plurality of affinity ligands having a range of binding specificities. An example of a useful affinity ligand expression library is the HuCAL Platinum.RTM. Platform (AbD Serotec, Bio-Rad), which provides phage display libraries of antibodies in Fab format representing an extensive array (>10.sup.10 members) of CDR sequence variability (Prassler, J., et al. (2011). "HuCAL PLATINUM, a synthetic Fab library optimized for sequence diversity and superior performance in mammalian expression systems." J. Mol. Biol. 413(1): 261-278). In some instances, the variability of the plurality of affinity ligands expressed by the library facilitate the identification of ligands that bind to a target with specific binding affinity under neutral buffer conditions and have substantially reduced specific binding under mild elution conditions as described in Section I.

[0070] To perform the method, a target of interest is linked or adsorbed to a solid support. The target may be such as a protein, nucleic acid, or other biomolecule as described above. The solid support may be the wall or floor of an assay vessel, or a dipstick or other implement to be inserted into an assay vessel, or particles (such as magnetic beads) placed inside or suspended in an assay vessel. Particles, and especially beads, can be useful in many embodiments, including beads that are microscopic in size (microparticles) and formed of a polymeric material.

[0071] To prevent non-specific binding of affinity ligands, the target-linked solid support may be blocked with a blocking buffer. For example, the blocking buffer may contain an animal protein blocker (such a milk or bovine serum albumin), a non-animal protein blocker (such as ChemiBLOCKER.TM.), or a detergent (such a Tween-20.RTM.). In some instances, the blocking buffer may contain a closely related antigen. For example, where the target is an IgM antibody with kappa light chain, the blocking buffer may contain an antibody of different isotype (e.g., IgG) with kappa light chain as a blocker. Inclusion of this blocker may help to avoid enrichment of affinity ligands that bind specifically to the kappa light chain or to heavy chain epitopes that are similar in IgM and IgG. In particular, where the target protein is an immunoglobulin and the plurality of affinity ligands are antibodies, the target-linked solid support may be blocked with one or more types of antibody light chains as a blocker. For example, where the ligand is human IgM or human IgA, the target-linked solid support may be blocked with human IgG1 lambda or IgG1 kappa as described in Examples 1 and 2. In some instances, the blocking buffer may contain culture media (for example, media that sustain growth of a eukaryotic cell culture producing immunoglobulin like IgE, IgA or IgM). Blocking with culture medium may be useful to avoid enrichment of affinity ligands with cross-reactivity to components in the culture medium. In some instances, this can be useful because the target proteins are expressed in cells that are grown in culture medium. For example, as described in Examples 1 and 2, where the ligand is human IgE, the target-linked solid support may be blocked with culture medium.

[0072] The expressed affinity ligands can be brought into contact with the target-linked solid support under neutral buffer conditions to achieve binding of the affinity ligands, if any ligands in the library have affinity for the target. To remove non-specifically or weakly bound affinity ligands, a washing step may be performed using neutral buffer conditions as described above in Section I.

[0073] An elution step may then be performed using the mild elution conditions as described above in Section I. Under such mild elution conditions, the binding of some of the affinity ligands to its target may be sufficiently reduced such that the affinity ligand and the target substantially dissociate from each other under these conditions. In one aspect, the mild elution conditions may comprise a pH of about 4.0 to about 5.5. In another aspect, the mild elution conditions may comprise about 1-2 M MgCl.sub.2.

[0074] The described methods allow for specific selection and enrichment of affinity ligands that specifically bind to a target under neutral buffer conditions and have substantially reduced specific binding to the target under mild elution conditions. Various elution conditions may be assessed to determine the optimal elution condition for a particular affinity ligand-target combination. Control elution conditions, in particular harsh elution conditions (such as pH 3.0), can be used for comparison purposes to assess the extent of elution obtained under the various elution conditions tested.

[0075] Additional assessment of affinity ligands identified using the methods may be performed. For example, once affinity ligands of interest are identified using the methods described above, the nucleotide sequences encoding the affinity ligands of interest may be cloned for larger scale production (for example, in vitro eukaryotic or bacterial expression). Cloned affinity ligands of interest may then be further screened to confirm specificity, such as, for example, by assessing binding affinity for other targets.

[0076] III. Affinity Chromatography Methods

[0077] Affinity ligands identified that specifically bind to a target under neutral buffer conditions and have substantially reduced binding to the target under mild elution conditions are useful for affinity chromatography. For example, such affinity ligands can be used for the affinity purification of targets in samples or expression cultures. The target may be a protein, nucleic acid, or other biological molecule. The affinity ligands are particularly useful for affinity chromatography for targets that are sensitive to harsh buffer conditions, including low pH (such as pH 3.0). For example, affinity chromatography using the affinity ligands described herein are useful for purification or assessment of targets that denature, dissociate into individual subunits, or dissociate from cofactors under harsh buffer conditions. Exemplary targets for the affinity chromatography methods described herein are immunoglobulins. As IgMs and IgAs are relatively sensitive to low pH conditions, affinity ligands that specifically bind to IgM or IgA are particularly useful for immune-chromatographic purification of IgMs and IgAs.

[0078] Affinity ligands for use in affinity chromatograph may be expressed and purified. These steps may be performed using conventional subcloning and expression technologies (for example, bacterial expression) using the nucleic acid sequences that encode these ligands or their binding domains. In some instances, the polynucleotides encoding the affinity ligands may be subcloned with an affinity tag or moiety that is useful for purification of the affinity ligands (for example, at least one of a FLAG.RTM. peptide, 6.times.-Histidine peptide, etc.). Affinity ligands are generally prepared in substantially purified form prior to use in generating an affinity chromatography matrix.

[0079] To perform affinity chromatography using the affinity ligands, the affinity ligands may be bound to a solid support to generate an affinity chromatography matrix. The affinity ligand is bound to or linked to the solid support. In some examples, the affinity ligand binds to the solid support through ionic interaction. In some instances, the affinity ligand is linked to the solid support through chemical bonds or cross-linking. Any solid support is contemplated for linkage to the affinity ligands. Various commercial matrices can be used to generate the affinity matrix with the affinity ligand bound thereto. Exemplary matrices include NHS-activated Sepharose matrix or Ni-NTA Agarose. The solid support may be selected based on characteristics of the purified affinity ligand, characteristics of the target, or intended uses of the target following affinity chromatography. The solid support can be, for example, porous or non-porous and can be in the form, for example, of a matrix, bead, particle, chip, or other conformation, for example, a membrane or a monolith (such as a single block, pellet, or slab of material). The solid support can be the wall or floor of an assay vessel, or a dipstick or other implement to be inserted into an assay vessel, or particles placed inside or suspended in an assay vessel. Particles, and especially beads, can be useful in many embodiments, including beads that are microscopic in size (microparticles) and formed of a polymeric material. Polymers useful as microparticles are those that are chemically inert relative to the components of the biological sample and to the assay reagents other than the affinity ligands that are immobilized on the microparticle surface. Examples of suitable polymers are polystyrenes, polyesters, polyethers, polyolefins, polyalkylene oxides, polyamides, polyurethanes, polysaccharides, celluloses, and polyisoprenes. Crosslinking is useful in many polymers for imparting structural integrity and rigidity to the microparticle. The size range of the microparticles can vary. In some embodiments, the microparticles range in diameter from about 0.3 micrometers to about 120 micrometers, and other embodiments, from about 0.5 micrometers to about 40 micrometers, and in still other embodiments, from about 2 micrometers to about 10 micrometers.

[0080] Affinity chromatography may be performed on a wide range of samples that contain the target of interest. In some instances, the samples are obtained from subjects (including humans, primates, non-primate mammals, birds, reptiles, and amphibians). For example, the sample may be a bodily fluid. The sample may also be cultured bacteria or other cells or tissues that express the target of interest, a cell culture supernatant, or a lysate from bacterial or eukaryotic cells. In addition, one or more purification steps may be performed on the sample to enrich the target prior to affinity chromatography using the affinity ligands described herein.

[0081] The sample is applied to the solid support to allow the target to be bound by the affinity ligand. The conditions for the binding may be neutral or physiological. To remove non-specifically or weakly bound non-target compounds, a washing step may be performed using neutral buffer conditions as described above in Section I.

[0082] To elute the target bound to the affinity ligand-solid support, an elution step is performed using mild elution conditions as described above in Section I. Under mild elution conditions, the binding of the affinity ligand to its target is reduced such that the affinity ligand and the target substantially dissociate from each other under these conditions. In one aspect, the mild elution conditions may comprise a pH of about 4.0 to about 5.5. In another aspect, the mild elution conditions may comprise about 1-2 M MgCl.sub.2. In some instances, elution may be performed as a single-step elution such that the target bound to the affinity ligand-solid support is eluted by exposing it to the mild elution conditions directly. In some instances, elution may be performed as a multiple-step elution such that the target bound to the affinity ligand-solid support is eluted by exposing it sequentially to multiple mild elution conditions having increasing salt concentrations or decreasing pH. In some instances, elution may be performed using a pH or salt gradient such that the target bound to the affinity ligand-solid support is eluted by exposing it to dynamic elution conditions as a gradient of linearly increasing salt concentration or decreasing pH.

[0083] The eluted target is useful for a variety of applications. For example, in the case that the target is an antibody (an immunoglobulin, such as IgM, IgA, or IgE), the eluted immunoglobulin target may be used, for example, as a therapeutic molecule, or as a diagnostic or laboratory reagent. In some instances, additional steps may be performed on the eluted target to prepare it for subsequent uses (for example, subsequent chromatography steps, filtration like ultrafiltration or diafiltration, dialysis, labeling, etc.).

[0084] IV. Kits

[0085] Kits containing affinity ligands that specifically bind to a target under neutral buffer conditions and elute the target under mild elution conditions are also contemplated. Kits may include one or more types of affinity ligands. The affinity ligands in the kit may be specific for the same target or for different targets. If the affinity ligands are specific for the same target, they may be specific for different epitopes of the target. In some instances, the affinity ligands are labeled (for example, with a peptide tag, or a chemical moiety for site-specific coupling to a matrix). The kits may include affinity ligands bound to a solid support. In some examples, the affinity ligand-bound solid support is packed into a chromatography column. Sometimes, the chromatography column is provided separately and the affinity ligand-bound solid support will be packed into the chromatography column prior to use. In some cases, the kit provides the affinity ligand and solid support separately along with instructions for coupling the affinity ligand to the solid support. The kit may further include an equilibration buffer, a washing buffer, an elution buffer, or some combination of these buffers. In some cases, more than one washing buffer or elution buffer is provided. Multiple elution buffers may be provided, with each elution buffer having a different elution condition (such as pH, salt type, or salt concentration). For example, different elution buffers may be provided for different affinity ligands included in the kit.

[0086] Exemplary buffers as referenced in this disclosure may include, for example, citrate, MES, or Bis-Tris, amongst others.

[0087] The foregoing description of certain embodiments, including illustrated embodiments, has been presented only for the purpose of illustration and description and is not intended to be exhaustive or to limit the disclosure to the precise forms disclosed. Numerous modifications, adaptations, and uses thereof will be apparent to those skilled in the art without departing from the scope of the disclosure. Certain features that are described in this specification in the context of separate embodiments can also be implemented in combination in a single implementation. Conversely, various features that are described in the context of a single implementation can also be implemented in multiple ways separately or in any suitable subcombination. Moreover, although features may be described above as acting in certain combinations, one or more features from a combination can in some cases be excised from the combination, and the combination may be directed to a subcombination or variation of a subcombination. Thus, particular embodiments have been described. Other embodiments are within the scope of the disclosure.

EXAMPLES

[0088] The following examples are offered to illustrate, but not to limit the claimed invention.

[0089] In the examples described below, antibodies were generated using the HuCAL PLATINUM.RTM. library that includes the CysDisplay.RTM. selection technology (Rothe, C., et al., 2008. "The human combinatorial antibody library HuCAL GOLD combines diversification of all six CDRs according to the natural immune system with a novel display method for efficient selection of high-affinity antibodies." J Mol Biol 376(4): 1182-1200.). The aim was to generate Fab antibodies against human IgM, IgA, and IgE that bind at neutral pH but can be eluted from the antigen under mild conditions (e.g. pH 4 to pH 5). Selection on the antigens using elution under mild conditions resulted in 14 antibodies against IgM, 40 antibodies against IgA, and 17 antibodies against IgE. Assays were performed to identify the antibodies with desirable properties for use as affinity chromatography reagent.

Example 1

[0090] This example provides a description of the materials and methods used in performing the experiments described in Example 2.

[0091] The antigens and closely related antigens (CRAs) are listed in Table 1 below.

TABLE-US-00001 TABLE 1 Antigens and CRAs Antigen/CRA Reference Source hIgM Ag05450 Human IgM, human plasma (AbD Serotec 5275-5504) hIgA Ag5451 Human IgA, human colostrum (AbD Serotec 5111-5504) hIgM Ag04815 Human IgM, human serum (Sigma I8260) hIgA Ag4813 Human IgA, human colostrum (Sigma I2636) hIgE Ag05681 Human IgE with lambda light chain, AbD00264 hIgE Ag05682 Human IgE with kappa light chain, (AbD Serotec, HCA190) hIgE Ag05711 Human IgE, myeloma (AbD Serotec PHP142) hIgG1lambda Ag05029 Human IgG1 lambda, human myeloma plasma (Sigma I5029) hIgG1lambda Ag05419 Human IgG1 lambda, human myeloma plasma (Sigma I5029) hIgG1kappa Ag05153 Human IgG1 kappa, human myeloma plasma (Sigma I5154)

[0092] Recombinant antibodies were isolated from the HuCAL PLATINUM.RTM. library of human antibody genes by three iterative rounds of panning with the antigens as described in Table 1, using standard protocols. (Knappik et al., J. Mol. Biol. 2000, 296:57-86; Prassler et al., J. Mol. Biol. 2011, 413:261-278; Krebs et al., J. Immunol. Methods 2001, 254:67-84; Jarutat et al., Biol. Chem. 2006, 387:995-1003.)

[0093] For pannings 274.17-274.22 (see Table 2), the antigens were passively adsorbed to microtiter plates (F96 Maxisorp.TM. Nunc-Immuno Plate #442404) for use in "solid phase panning" as described below.

[0094] For pannings 274.4-274.9 and 289.12-14, the antigens were coupled to Dynal M-450 Epoxy beads (Invitrogen 140-11). The antigen coupled beads were incubated overnight at room temperature, blocked by addition of Tris, pH7.4, and subsequently re-suspended in PBS.

[0095] The phage antibody library was incubated with blocking buffer containing the CRAs as set forth in Table 1 and Table 2. The blocked library was then incubated with the antigen coupled beads, and non-specific or blocked antibodies were washed off. Specific antibody phage were eluted as noted in Table 2, by incubation with pH 3 elution buffer as control selection (150 mM NaCl, 100 mM Citrate buffer pH 3), with pH 4 elution buffer (150 mM NaCl, 100 mM Citrate buffer pH 4), or pH 5 elution buffer (150 mM NaCl, 100 mM Citrate buffer or 150 mM NaCl, 100 mM Sodium Acetate buffer) for the pH elution conditions or with high salt elution buffer (2M MgCl.sub.2 in PBS). The pH elution condition buffers were neutralized with 1M Tris before E. coli infection. Phagemid containing bacteria were grown overnight at 37.degree. C. and new antibody displaying phage were produced for the next panning round.

[0096] After 3 rounds of panning, the enriched pool of Fab genes was isolated and inserted into the E. coli expression vector pMx11-FH that leads to functional periplasmic expression of monovalent Fab fragments. (Rauchenberger et al., J. Biol. Chem. 2003, 278:38194-38205.) Each Fab includes a FLAG tag and a 6.times.His tag in tandem at the C-terminus of the heavy chain.

[0097] E. coli TG1F (TG1 depleted for the F pilus; Rauchenberger et al. 2003) was then transformed with the ligated expression vectors. Following transformation, 368 individual colonies were randomly picked for each panning and grown in microtiter plates, which were then stored with 15% glycerol at -80.degree. C. These master plates were replicated and the resulting daughter plates were used for expression of the antibodies. After induction of antibody expression with 1 mM Isopropyl-.beta.-D-thiogalactopyranosid (IPTG) overnight at 22.degree. C., the cultures were chemically lysed, and the crude extracts were tested in Enzyme-Linked Immunosorbent Assay (ELISA) with the immobilized antigens and closely related antigens for the presence of antibody fragments that bind specifically to the immobilized antigens. In addition binding to IgM or IgA from a different source (Sigma) was tested. These antigens were also used to determine which antibodies can be washed of the ELISA plate after incubation with the different elution buffers for 20 min. The sequences of the antibody VH and VL complementarity-determining regions (CDR) were determined from a selection of the clones that gave a strong signal on the antigens in the ELISA (at least 5-fold above the background signal, antigens from AbD Serotec and Sigma) and a weak or no signal on the CRAs and on the antigen after elution buffer treatment (below 5-fold above the background signal). Clones containing antibodies with unique sequence were chosen for antibody production.

TABLE-US-00002 TABLE 2 Panning, Blocking and Screening Antigens Pan- Panning Elution Primary ELISA code Antigens Strategy CRAs Used for Blocking Screening on Antigens 274.4 hIgM pH 3 Elution Ag05029 & Ag05153 Ag05450 (hIgM) (Ag05450) (hIgG1 lambda & kappa) to Ag05029 (hIgG1lambda) final conc. of 50 .mu.g/mL Ag05153 (hIgG1kappa) 100 .mu.l cell culture medium Ag04815 (hIgM, Sigma) Ag04815 (elution buffer incubation) 274.5 hIgA Bead Ag05029 & Ag05153 Ag05451 (hIgA) (Ag05451) Panning (hIgG1 lambda & kappa) to Ag05029 (hIgG1lambda) pH 3 Elution final conc. of 50 .mu.g/mL Ag05153 (hIgG1kappa) 100 .mu.l cell culture medium Ag04813 (hIgA, Sigma) Ag04813 (elution buffer incubation) 274.6 hIgM Bead Ag05029 & Ag05153 Ag05450 (hIgM) (Ag05450) Panning (hIgG1 lambda & kappa) to Ag05029 (hIgG1lambda) pH 4 Elution final conc. of 50 .mu.g/mL Ag05153 (hIgG1kappa) 100 .mu.l cell culture medium Ag04815 (hIgM, Sigma) Ag04815 (elution buffer incubation) 274.7 hIgA Bead Ag05029 & Ag05153 Ag05451 (hIgA) (Ag05451) Panning (hIgG1 lambda & kappa) to Ag05029 (hIgG1lambda) pH 4 Elution final conc. of 50 .mu.g/mL Ag05153 (hIgG1kappa) 100 .mu.l cell culture medium Ag04813 (hIgA, Sigma) Ag04813 (elution buffer incubation) 274.8 hIgM Bead Ag05029 & Ag05153 Ag05450 (hIgM) (Ag05450) Panning (hIgG1 lambda & kappa) to Ag05029 (hIgG1lambda) High Salt final conc. of 50 .mu.g/mL Ag05153 (hIgG1kappa) Elution (2M 100 .mu.l cell culture medium Ag04815 (hIgM, Sigma) MgCl.sub.2) Ag04815 (elution buffer incubation) 274.9 hIgA Bead Ag05029 & Ag05153 Ag05451 (hIgA) (Ag05451) Panning (hIgG1 lambda & kappa) to Ag05029 (hIgG1lambda) High Salt final conc. of 50 .mu.g/mL Ag05153 (hIgG1kappa) Elution (2M 100 .mu.l cell culture medium Ag04813 (hIgA, Sigma) MgCl.sub.2) Ag04813 (elution buffer incubation) 274.17 hIgM Bead Ag05029 & Ag05153 Ag05450 (hIgM) (Ag05450) Panning (hIgG1 lambda & kappa) to Ag05029 (hIgG1lambda) pH 3 Elution final conc. of 50 .mu.g/mL Ag05153 (hIgG1kappa) 100 .mu.l cell culture medium Ag04815 (hIgM, Sigma) Ag04815 (elution buffer incubation) 274.18 hIgA Bead Ag05029 & Ag05153 Ag05451 (hIgA) (Ag05451) Panning (hIgG1 lambda & kappa) to Ag05029 (hIgG1lambda) pH 3 Elution final conc. of 50 .mu.g/mL Ag05153 (hIgG1kappa) 100 .mu.l cell culture medium Ag04813 (hIgA, Sigma) Ag04813 (elution buffer incubation) 274.19 hIgM Bead Ag05029 & Ag05153 Ag05450 (hIgM) (Ag05450) Panning (hIgG1 lambda & kappa) to Ag05029 (hIgG1lambda) pH 4 Elution final conc. of 50 .mu.g/mL Ag05153 (hIgG1kappa) 100 .mu.l cell culture medium Ag04815 (hIgM, Sigma) Ag04815 (elution buffer incubation) 274.20 hIgA Bead Ag05029 & Ag05153 Ag05451 (hIgA) (Ag05451) Panning (hIgG1 lambda & kappa) to Ag05029 (hIgG1lambda) pH 4 Elution final conc. of 50 .mu.g/mL Ag05153 (hIgG1kappa) 100 .mu.l cell culture medium Ag04813 (hIgA, Sigma) Ag04813 (elution buffer incubation) 274.21 hIgM Bead Ag05029 & Ag05153 Ag05450 (hIgM) (Ag05450) Panning (hIgG1 lambda & kappa) to Ag05029 (hIgG1lambda) High Salt final conc. of 50 .mu.g/mL Ag05153 (hIgG1kappa) Elution (2M 100 .mu.l cell culture medium Ag04815 (hIgM, Sigma) MgCl.sub.2) Ag04815 (elution buffer incubation) 274.22 hIgA Bead Ag05029 & Ag05153 Ag05451 (hIgA) (Ag05451) Panning (hIgG1 lambda & kappa) to Ag05029 (hIgG1lambda) High Salt final conc. of 50 .mu.g/mL Ag05153 (hIgG1kappa) Elution (2M 100 .mu.l cell culture medium Ag04813 (hIgA, Sigma) MgCl.sub.2) Ag04813 (elution buffer incubation) 289.12 hIgE Bead Ag05419 & Ag05153 Ag05681 (hIgE/lambda) lambda Panning (hIgG1 lambda & kappa) to Ag05682 (hIgE/kappa) (Ag05681) pH 4 final conc. of 50 .mu.g/mL Ag05419 (hIgG1lambda) hIgE/kappa Elution, 100 .mu.l cell culture medium Ag05153 (hIgG1kappa) (Ag05682) 10 min Ag05681 (elution buffer Elution Step incubation) 289.13 hIgE Bead Ag05419 & Ag05153 Ag05681 (hIgE/lambda) lambda Panning (hIgG1 lambda & kappa) to Ag05682 (hIgE/kappa) (Ag05681) pH 4 final conc. of 50 .mu.g/mL Ag05419 (hIgG1lambda) hIgE/kappa Elution, 100 .mu.l cell culture medium Ag05153 (hIgG1kappa) (Ag05682) 5 min Ag05681 (elution buffer Elution Step incubation) 289.14 hIgE Bead Ag05419 & Ag05153 Ag05681 (hIgE/lambda) lambda Panning (hIgG1 lambda & kappa) to Ag05682 (hIgE/kappa) (Ag05681) pH 5 final conc. of 50 .mu.g/mL Ag05419 (hIgG1lambda) hIgE/kappa Elution, 100 .mu.l cell culture medium Ag05153 (hIgG1kappa) (Ag05682) 10 min Ag05681 (elution buffer Elution Step incubation)

[0098] The ELISA screening protocol is set forth in the Table 3 below.

TABLE-US-00003 TABLE 3 ELISA Screening Protocol Plates 384 well Maxisorp microtiter plates (MTP), black, flat bottom, Polystyrene (Nunc 460518) Coating 20 .mu.L/well of antigens at 5 .mu.g/mL in PBS pH 7.4; overnight (o/n) incubation at 4.degree. C. Wash 2x PBST (PBS with 0.05% Tween .RTM. 20) Blocking 100 .mu.L of 5% non-fat dried milk in PBST for 1-2 h at room temperature (RT) Wash 2x PBST Primary Ab 20 .mu.L/well of crude E. coli lysate of expression (HuCAL .RTM.-Fab) culture containing HuCAL .RTM.-Fab (pre-blocked with a final concentration of 5% non-fat dried milk in PBST), 1 h at RT Wash 5x PBST Secondary Ab 20 .mu.L/well of anti-His tag, HRP conjugate (Qiagen 34460), 1:2000 dilution in HiSpec buffer (AbD Serotec BUF049), 1 h at RT Wash 5x PBST Detection 20 .mu.L/well QuantaBlu .RTM. (Thermo Scientific 15169) Reader Settings Excitation at 320 .+-. 25 nm, emission at 420 .+-. 35 nm

[0099] E. coli TG1F.sup.- cultures (250 mL) containing the chosen antibody genes were grown at 30.degree. C. until OD.sub.600nm reached 0.5, and the antibody expression was induced by adding IPTG to a final concentration of 1 mM. After further incubation for at least 14 hours at 30.degree. C., the cells were harvested, chemically lysed, and the soluble crude extract was subjected to one-step affinity chromatography (Ni-NTA Agarose, Qiagen 1018240). After elution of the purified antibodies from the column, the buffer was changed from elution buffer to 3.times.PBS, pH 7.4, and the concentration was determined by UV280 nm measurement. Purity and activity was tested subsequently by Coomassie-stained SDS-PAGE and ELISA, respectively.

[0100] The quality control ELISA (indirect ELISA) protocol using purified HuCAL.RTM. antibodies is set forth in Table 4 below.

TABLE-US-00004 TABLE 4 QC ELISA Protocol Plate 384 well Maxisorp MTP, black, flat bottom, Polystyrene (Nunc 460518) Coating 20 .mu.L/well of antigen at 5 .mu.g/mL in PBS, o/n at 4.degree. C. Wash 2x PBST (PBS with 0.05% Tween-20 .RTM.) Blocking 100 .mu.L of 5% non-fat dried milk in PBST for 1-2 hr at RT Wash 2x PBST Primary Ab 20 .mu.L/well of antibody at 2 .mu.g/mL in PBST for 1 hr at (HuCAL .RTM.-Fab) RT Wash 5x PBST Secondary Ab 20 .mu.L/well of anti-His tag, HRP conjugate (Qiagen 34460), 1:2000 dilution in HiSpec buffer (AbD Serotec BUF049), 1 hr at RT Wash 5x PBST Optional: manually wash specific wells with elution buffer, incubate for 5 min, remove buffer, repeat 2x Detection 20 .mu.L/well QuantaBlu .RTM. (Thermo Scientific 15169) Reader Settings Excitation at 320 .+-. 25 nm, emission at 420 .+-. 35 nm Background Signal values on unspecific antigens (BSA, GST) were used for the calculation of background signal

Example 2

[0101] The results of the methods described in Example 1 are described in this example.

Primary ELISA Screening

[0102] The outcome of a panning is tested in the primary screening on several antigens and closely related antigens (CRA). The antigen role defines whether positive (+) signals are expected (positive controls for the panning antigen) or whether no signal (-) is wanted (negative controls; CRAs).

[0103] An ELISA signal on an antigen plate (positive control) is considered a hit if the signal is 5-fold above background. Background is the average value of a number of wells on the plate that were not treated with antigen or antibody.

[0104] A signal on a CRA (negative control) is counted, and during the analysis subtracted from the positive hits, if the signal is at least 2-fold above background.

[0105] A clone is considered a hit if it is positive on all antigens (positive controls) and negative on all CRAs (negative controls).

[0106] The values in Table 5 indicate the numbers of hits according to the above definition. The Analysis column indicates the number of hits that no longer bind to the antigen under the test elution conditions.

TABLE-US-00005 TABLE 5 Primary screening ELISA results Antigen Role + + - - - hIgG1 hIgG1 hIgM, hIgM, hIgM, lambda, kappa, Ag04815 & Panning Ag05450 Ag04815 Ag05029 Ag05153 elution buffer Analysis 274.4 234 43 0 2 11, pH 3 38 274.6 191 59 1 2 5, pH 4 55 274.8 234 163 3 2 7, high salt 153 274.17 239 8 0 15 1, pH 3 4 274.19 228 106 2 0 1, pH 4 93 274.21 28 0 1 1 7, high salt 0 hIgG1 hIgG1 hIgA, hIgA, hIgA, lambda, kappa, Ag04813 & Ag05451 Ag04813 Ag05029 Ag05153 elution buffer Analysis 274.5 205 200 2 1 71, pH 3 125 274.7 223 201 1 31 96, pH 4 94 274.9 285 286 1 8 271, high salt 5 274.18 193 215 3 1 52, pH 3 132 274.20 173 184 0 6 119, pH 4 58 274.22 174 220 6 1 166, high salt 16 hIgG1 hIgG1 hIgE, hIgE, hIgE, lambda, kappa, Ag05681 & Ag05681 Ag05682 Ag05419 Ag05153 elution buffer Analysis 289.12 9 1 0 3 4, pH 4 1 289.13 20 24 24 164 208, pH 4, 9 not used* 289.14 25 186 147 10 21, pH 5 10 *The number of hits was too high, so this result was considered to be an artifact and was not used to pick clones for further characterization.

Sequencing and Identification of Unique Antibodies

[0107] For the IgE project, 20 clones (289.12: 1, 289.13: 9, 289.14: 10) were sequenced and resulted in 17 different antibodies (AbD22512, AbD22628-22643). The heavy and light chain CDR1, CDR2, and CDR3 region sequences of these antibodies are shown in FIG. 1A and FIG. 1B.

[0108] For the IgA project, a total of 45 clones (274.7: 18, 274.9: 3, 274.20: 15, 274.22: 9) were selected for further analysis. Sequencing of the gene regions coding for VH and VL revealed 40 different antibodies (AbD20776-20791, AbD20797-20799, AbD20801-20812, AbD20813-20821). The heavy and light chain CDR1, CDR2, and CDR3 region sequences of these antibodies are shown in FIG. 2A and FIG. 2B.

[0109] For the IgM project, a total of 50 clones (274.6: 15, 274.8: 20, 274.19: 15) were selected for further analysis. Sequencing of the gene regions coding for VH and VL revealed 14 different antibodies (AbD20768-20775, AbD20792-20796, AbD20800). The heavy and light chain CDR1, CDR2, and CDR3 region sequences of these antibodies are shown in FIG. 3A FIG. 3B; full heavy and light chain sequences for certain of these antibodies is shown in FIGS. 4A-4I.

[0110] The antibodies were expressed, purified via affinity chromatography and tested using ELISA. The results are shown in FIGS. 5-9 and in Tables 6-8 and described further below.

[0111] For each clone shown, a number of antigens and conditions were tested. The bars indicate the signal strength plotted as specific fluorescence divided by background fluorescence. The designation of the clones is "clone-name.batch-number." For example, AbD20768.1 is the first batch of AbD20768

[0112] FIG. 5: BSA and N1-CD33-6.times.His are unrelated antigens. hIgG1Kap-ctrl and hIgG1lambdaCtrl are CRAs (human IgG1 isotype with kappa and lambda light chain, respectively). hIgM is human IgM from AbD Serotec (5275-5504). H-IgM (Sigma) is human IgM from Sigma (18260). Antigen with "pH4 wash" indicate the residual signal on that antigen after 3 consecutive 5 minute incubations of the corresponding wells with the elution buffer (pH 4).

[0113] FIG. 6: BSA and N1-CD33-His6 are unrelated antigens. hIgG1Kap-ctrl and hIgG1lambdaCtrl are CRAs (human IgG1 isotype with kappa and lambda light chain, respectively). hIgM is human IgM from AbD Serotec (5275-5504). H-IgM (Sigma) is human IgM from Sigma (18260). Antigen with "2M MgCl.sub.2 wash" indicate the residual signal on that antigen after 3 consecutive 5 minute incubations of the corresponding wells with the elution buffer (2M MgCl.sub.2 in PBS).

[0114] FIG. 7: BSA and GST are unrelated antigens. hIgG1Kap-ctrl and hIgG1lambdaCtrl are CRAs (human IgG1 isotype with kappa and lambda light chain, respectively). hIgA is human IgA from AbD Serotec (5111-5504). HIgA (Sigma) is human IgA from Sigma (12636). Antigen with "pH4 wash" indicate the residual signal on that antigen after 3 consecutive 5 minute incubations of the corresponding wells with the elution buffer (pH 4). A number of antibodies that were originally positive in the primary screening turned out to be negative here (AbD20801 to AbD20812).

[0115] FIG. 8: BSA and N1-CD33-His6 are unrelated antigens. hIgG1Kap-ctrl and hIgG1lambdaCtrl are CRAs (human IgG1 isotype with kappa and lambda light chain, respectively). hIgA is human IgA from AbD Serotec (5111-5504). HIgA (Sigma) is human IgA from Sigma (12636). Antigen with "2M MgCl.sub.2 wash" indicate the residual signal on that antigen after 3 consecutive 5 minute incubations of the corresponding wells with the elution buffer (2M MgCl.sub.2 in PBS). A number of antibodies that were originally positive in the primary screening turned out to be negative here (AbD20814-AbD20821).

[0116] FIG. 9: BSA and GST are unrelated antigens. hIgG1Kap-ctrl and hIgG1lambdaCtrl are CRAs (human IgG1 isotype with kappa and lambda light chain, respectively). hIgG4 kappa is human IgG4 with kappa light chain (Sigma 14639). hIgM is human IgM from AbD Serotec (5275-5504). AbD00264_hIgG1f is a human antibody derived from HuCAL and formatted into the IgG1 isotype. AbD00264_hIgE is the same antibody formatted into the IgE isotype. AbD18705_hIgE is another HuCAL-derived antibody formatted into the IgE isotype. hIgE is human IgE from myeloma (AbD Serotec PHP 142).

TABLE-US-00006 TABLE 6 Overview of Specific Anti-hIgM Antibodies Antigen Elution Antibody Antigen Number Name conc. [mg/ml] buffer AbD20768.1 Ag05450 hIgM 1.56 pH 4 AbD20769.1 Ag05450 hIgM 1.65 pH 4 AbD20770.1 Ag05450 hIgM 1.6 pH 4 AbD20771.1 Ag05450 hIgM 1.7 pH 4 AbD20772.1 Ag05450 hIgM 0.88 pH 4 AbD20773.1 Ag05450 hIgM 0.82 pH 4 AbD20774.1 Ag05450 hIgM 1.48 pH 4 AbD20775.1 Ag05450 hIgM 1.11 pH 4 AbD20800.1 Ag05450 hIgM 0.72 pH 4 AbD20792.1 Ag05450 hIgM 0.97 2M MgCl.sub.2 AbD20793.1 Ag05450 hIgM 0.3 2M MgCl.sub.2 AbD20794.1 Ag05450 hIgM 0.93 2M MgCl.sub.2 AbD20795.1 Ag05450 hIgM 0.73 2M MgCl.sub.2 AbD20796.1 Ag05450 hIgM 0.93 2M MgCl.sub.2

TABLE-US-00007 TABLE 7 Overview of Specific Anti-hIgA Antibodies Antigen conc. Antibody Number Antigen Name [mg/ml] Elution buffer AbD20776.1 Ag05451 hIgA 1.13 pH 4 AbD20777.1 Ag05451 hIgA 0.96 pH 4 AbD20778.1 Ag05451 hIgA 1.32 pH 4 AbD20779.1 Ag05451 hIgA 1.38 pH 4 AbD20780.1 Ag05451 hIgA 1.54 pH 4 AbD20781.1 Ag05451 hIgA 0.99 pH 4 AbD20782.1 Ag05451 hIgA 0.99 pH 4 AbD20783.1 Ag05451 hIgA 1.25 pH 4 AbD20784.1 Ag05451 hIgA 1.03 pH 4 AbD20785.1 Ag05451 hIgA 1.46 pH 4 AbD20786.1 Ag05451 hIgA 1.22 pH 4 AbD20787.1 Ag05451 hIgA 1.35 pH 4 AbD20788.1 Ag05451 hIgA 1.5 pH 4 AbD20789.1 Ag05451 hIgA 1.68 pH 4 AbD20790.1 Ag05451 hIgA 1.61 pH 4 AbD20791.1 Ag05451 hIgA 1.25 pH 4 AbD20797.1 Ag05451 hIgA 1.43 2M MgCl.sub.2 AbD20798.1 Ag05451 hIgA 1.43 2M MgCl.sub.2 AbD20799.1 Ag05451 hIgA 1.04 2M MgCl.sub.2 AbD20813.1 Ag05451 hIgA 1.21 2M MgCl.sub.2

TABLE-US-00008 TABLE 8 Overview of Specific Anti-hIgE Antibodies Antigen Antigen conc. Elution Antibody Number Name [mg/ml] buffer AbD22512.1 Ag05681 hIgE 1.76 pH 4 AbD22628.1 Ag05681 hIgE 1.41 pH 4 AbD22629.1 Ag05681 hIgE 1.83 pH 4 AbD22630.1 Ag05681 hIgE 1.78 pH 4 AbD22631.1 Ag05681 hIgE 1.75 pH 4 AbD22632.1 Ag05681 hIgE 1.77 pH 4 AbD22633.1 Ag05681 hIgE 1.59 pH 4 AbD22634.1 Ag05681 hIgE 0.69 pH 4 AbD22635.1 Ag05681 hIgE 1.72 pH 5 AbD22636.1 Ag05681 hIgE 1.70 pH 5 AbD22637.1 Ag05681 hIgE 1.20 pH 5 AbD22638.1 Ag05681 hIgE 1.70 pH 5 AbD22639.1 Ag05681 hIgE 1.37 pH 5 AbD22640.1 Ag05681 hIgE 1.58 pH 5 AbD22641.1 Ag05681 hIgE 1.76 pH 5 AbD22642.1 Ag05681 hIgE 0.90 pH 5 AbD22643.1 Ag05681 hIgE 1.69 pH 5

Example 3

[0117] Selected antibody Fab fragments isolated as described in Example 2 were used as affinity ligands to purify an immunoglobulin target. Two anti-IgM antibody Fab fragments were selected as representative affinity ligands: AbD20771 and AbD20775. The affinity ligands were expressed in E. coli as monomers having a FLAG-His6 tag on the heavy chain. The ligands were purified using Ni-NTA agarose.

[0118] Four hIgM molecules were selected as exemplary target molecules to assess the purification capability of the ligands. These ligands are set forth in Table 9. Supernatant containing these ligands was collected during culturing of the cell lines noted in Table 9. OBT1524 hIgM (AbD Serotec, Bio-Rad), an IgM protein purified from myeloma serum, was used as a standard control.

TABLE-US-00009 TABLE 9 IgM Target Molecules For Purification Target Molecule Cell Line Expression AbD00264 hIgM lambda (AbD Expression in a stable human HKB11 Serotec, BioRad) cell pool (AbD Serotec, BioRad) AbD18705 hIgM kappa (AbD Expression in a stable human HKB11 Serotec, BioRad) cell pool (AbD Serotec, BioRad) AbD18777 hIgM kappa (AbD Transient expression in human HKB11 Serotec, BioRad) cell line (AbD Serotec, BioRad)

[0119] The purified affinity ligands were coupled to 1 ml "HiTrap NHS-activated HP" columns according to manufacture's protocol (GE Healthcare #17-0716-01), with 7 mg of the ligand being applied to the column. The columns were run on a GE AKTAxpress.TM. FPLC instrument. The supernatant sample (200 ml) was loaded using a flow rate of 0.4 ml/min (62 cm/h; retention time of 2.4 min) using PBS (pH about 7.0-7.2) as binding and washing buffer. The elution buffer was 100 mM citrate, 150 mM NaCl, pH 4.0. Following elution, a neutralization buffer (1 M Tris/HCl pH 9.0) was used to neutralize the pH of the purified IgM targets.

[0120] Exemplary results are shown for target molecule AbD18705 IgM. Results for the other two target molecules were similar. Elution profiles (UV 280 nm) are shown in FIG. 10 (collected fractions are indicated). The AbD18705 IgM target molecule is captured from cell culture supernatant and eluted under mild conditions (pH 4.0) using both affinity ligand AbD20771 and AbD20775.

[0121] To assess the purity and integrity of the purified target molecules, the purified protein fractions (A3/4/5 for the AbD20775 column and A10/11/12 for the AbD20771 column, as identified in FIG. 10; 1 .mu.g protein/lane) were run on 4-20% Mini-PROTEAN.TM. Stain-free TGX gels (Bio-Rad). Exemplary reducing and non-reducing lanes are shown for each fraction, respectively, in FIG. 11. Comparisons were made to the purified OBT1524 IgM standard control protein (FIG. 11, right panel). The purity of the AbD18705 IgM target molecule in the fractions is high under reducing conditions and both heavy and light chains can be detected. Under non-reducing conditions, assembled IgM is detectable.

[0122] Size exclusion chromatography (SEC) using a Superose 6 SEC column (GE Healthcare) was also performed to assess the integrity of the purified target molecules. FIG. 12 shows an overlay of SEC runs (UV280 nm signal) for fractions A4/5/10/11/12 as identified in FIG. 10. The peak retention volume was 10.8 ml, corresponding to a MW of 916 kDa (calculated MW for IgM AbD18705 in pentameric form without glycosylation is 859 kDa. All fractions show an identical elution behaviour indicating that assembled, non-aggregated IgM is present in the fractions. No degradation or aggregation is visible.

[0123] Finally, ELISA analysis of the various fractions was performed to assess activity and specificity of the purified AbD18705 hIgM target molecule. Negative control antigens (BSA, N1-CD33-6.times.His, GST) were coated at 5 .mu.g/ml onto the ELISA plate along with the specific antigen His-GFP, which is the antigen of AbD18705. The six fractions A3/4/5 and A10/11/12 of the purified IgM AbD18705, numbered 1 to 6 here, were added (20 .mu.l each) after washing, blocking and additional washing. Detection was performed using an anti-human IgM HRP conjugate (AbD Serotec) in combination with Quantablu.TM. substrate. As shown in FIG. 13, the purified AbD18705 hIgM antibody fractions all recognize the His-GFP antigen specifically and, thus, have a native active conformation.

[0124] All patents, patent publications, patent applications, journal articles, books, technical references, and the like discussed in the instant disclosure are incorporated herein by reference in their entirety for all purposes.

[0125] It is to be understood that the figures and descriptions of the invention have been simplified to illustrate elements that are relevant for a clear understanding of the invention. It should be appreciated that the figures are presented for illustrative purposes and not as construction drawings. Omitted details and modifications or alternative embodiments are within the purview of persons of ordinary skill in the art.

[0126] It can be appreciated that, in certain aspects of the invention, a single component may be replaced by multiple components, and multiple components may be replaced by a single component, to provide an element or structure or to perform a given function or functions. Except where such substitution would not be operative to practice certain embodiments of the invention, such substitution is considered within the scope of the invention.

[0127] The examples presented herein are intended to illustrate potential and specific implementations of the invention. It can be appreciated that the examples are intended primarily for purposes of illustration of the invention for those skilled in the art. There may be variations to these diagrams or the operations described herein without departing from the spirit of the invention. For instance, in certain cases, method steps or operations may be performed or executed in differing order, or operations may be added, deleted or modified.

[0128] Different arrangements of the components depicted in the drawings or described above, as well as components and steps not shown or described are possible. Similarly, some features and sub-combinations are useful and may be employed without reference to other features and sub-combinations. Embodiments of the invention have been described for illustrative and not restrictive purposes, and alternative embodiments will become apparent to readers of this patent. Accordingly, the present invention is not limited to the embodiments described above or depicted in the drawings, and various embodiments and modifications can be made without departing from the scope of the claims below.

Sequence CWU 1

1

288110PRTArtificial Sequencesynthetic affinity ligand peptide sequence 1Gly Tyr Ser Phe Ser Ser Tyr Trp Ile Thr1 5 10210PRTArtificial Sequencesynthetic affinity ligand peptide sequence 2Gly Tyr Thr Phe Thr Gly Tyr Asp Ile His1 5 10310PRTArtificial Sequencesynthetic affinity ligand peptide sequence 3Gly Gly Thr Phe Ser Asp Tyr Ala Ile Ser1 5 10410PRTArtificial Sequencesynthetic affinity ligand peptide sequence 4Gly Gly Thr Phe Ser Thr Tyr Ala Ile Ser1 5 10510PRTArtificial Sequencesynthetic affinity ligand peptide sequence 5Gly Tyr Ser Phe Thr Ser Tyr Trp Ile Gly1 5 10612PRTArtificial Sequencesynthetic affinity ligand peptide sequence 6Gly Asp Ser Val Ser Arg Asn Ser Ala Ala Trp Asn1 5 10710PRTArtificial Sequencesynthetic affinity ligand peptide sequence 7Gly Gly Thr Phe Ser Ser Tyr Tyr Ile His1 5 10810PRTArtificial Sequencesynthetic affinity ligand peptide sequence 8Gly Phe Thr Phe Ser Ser Tyr Val Met Thr1 5 10910PRTArtificial Sequencesynthetic affinity ligand peptide sequence 9Gly Gly Thr Phe Asn Ser Tyr Ala Ile His1 5 101010PRTArtificial Sequencesynthetic affinity ligand peptide sequence 10Gly Gly Thr Phe Ser Asp Tyr Ala Ile Ser1 5 101110PRTArtificial Sequencesynthetic affinity ligand peptide sequence 11Gly Phe Thr Phe Ser Ser Tyr Ala Ile Ser1 5 101210PRTArtificial Sequencesynthetic affinity ligand peptide sequence 12Gly Gly Thr Phe Ser Asp Tyr Ala Ile Ser1 5 101310PRTArtificial Sequencesynthetic affinity ligand peptide sequence 13Gly Phe Thr Phe Ser Thr Tyr Ala Met His1 5 101410PRTArtificial Sequencesynthetic affinity ligand peptide sequence 14Gly Phe Thr Phe Arg Ser His Gly Met Ser1 5 101510PRTArtificial Sequencesynthetic affinity ligand peptide sequence 15Gly Phe Thr Phe Arg Ser His Gly Met Ser1 5 101610PRTArtificial Sequencesynthetic affinity ligand peptide sequence 16Gly Tyr Thr Phe Thr Gly Tyr Tyr Met Ser1 5 101710PRTArtificial Sequencesynthetic affinity ligand peptide sequence 17Gly Phe Thr Phe Ser Asp Tyr Ala Ile His1 5 101820PRTArtificial Sequencesynthetic affinity ligand peptide sequence 18Trp Met Gly Thr Ile Phe Pro Asp Asp Ser Tyr Thr Ile Tyr Ser Pro1 5 10 15Ser Phe Gln Gly 201920PRTArtificial Sequencesynthetic affinity ligand peptide sequence 19Trp Met Gly Trp Ile Ala Pro Tyr Asn Gly Gly Thr Asn Tyr Ala Gln1 5 10 15Lys Phe Gln Gly 202020PRTArtificial Sequencesynthetic affinity ligand peptide sequence 20Trp Met Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln1 5 10 15Lys Phe Gln Gly 202120PRTArtificial Sequencesynthetic affinity ligand peptide sequence 21Trp Met Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asp Tyr Ala Gln1 5 10 15Lys Phe Gln Gly 202220PRTArtificial Sequencesynthetic affinity ligand peptide sequence 22Trp Met Gly Ile Ile Asp Pro Ser Asn Ser Asp Thr Arg Tyr Ser Pro1 5 10 15Ser Phe Gln Gly 202321PRTArtificial Sequencesynthetic affinity ligand peptide sequence 23Trp Leu Gly Arg Ile Gln Tyr Arg Ser Lys Trp Ile Asn Asp Tyr Ala1 5 10 15Val Ser Val Lys Ser 202420PRTArtificial Sequencesynthetic affinity ligand peptide sequence 24Trp Met Gly Gly Ile Gly Pro Ile Phe Gly Val Ala Asn Tyr Ala Gln1 5 10 15Lys Phe Gln Gly 202520PRTArtificial Sequencesynthetic affinity ligand peptide sequence 25Trp Val Ser Ala Ile Ser Tyr Asp Gly Ser Ser Thr Tyr Tyr Ala Asp1 5 10 15Ser Val Lys Gly 202620PRTArtificial Sequencesynthetic affinity ligand peptide sequence 26Trp Met Gly Gly Ile Ala Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln1 5 10 15Lys Phe Gln Gly 202720PRTArtificial Sequencesynthetic affinity ligand peptide sequence 27Trp Met Gly Gly Ile Glu Pro Val Phe Gly Thr Ala Lys Tyr Ala Gln1 5 10 15Lys Phe Gln Gly 202820PRTArtificial Sequencesynthetic affinity ligand peptide sequence 28Trp Val Ser Tyr Ile Ser Ser Gly Gly Ser Glu Thr Tyr Tyr Ala Asp1 5 10 15Ser Val Lys Gly 202920PRTArtificial Sequencesynthetic affinity ligand peptide sequence 29Trp Met Gly Gly Ile Ser Pro Asp Phe Gly Thr Ala Asn Tyr Ala Gln1 5 10 15Lys Phe Gln Gly 203022PRTArtificial Sequencesynthetic affinity ligand peptide sequence 30Trp Val Gly Arg Ile Lys Ser Lys Gln Asp Gly Gly Thr Thr Asp Tyr1 5 10 15Ala Ala Pro Val Lys Gly 203120PRTArtificial Sequencesynthetic affinity ligand peptide sequence 31Trp Val Ser Thr Ile Ser Gly Ser Gly Ser Asn Thr Tyr Tyr Ala Asp1 5 10 15Ser Val Lys Gly 203220PRTArtificial Sequencesynthetic affinity ligand peptide sequence 32Trp Val Ser Thr Ile Ser Gly Ser Gly Ser Asn Thr Tyr Tyr Ala Asp1 5 10 15Ser Val Lys Gly 203320PRTArtificial Sequencesynthetic affinity ligand peptide sequence 33Trp Met Gly Tyr Ile Ser Pro Tyr Ser Gly Lys Thr Asn Tyr Ala Gln1 5 10 15Lys Phe Gln Gly 203422PRTArtificial Sequencesynthetic affinity ligand peptide sequence 34Trp Val Gly Arg Ile Lys Ser His Ala Tyr Gly Gly Thr Thr Asp Tyr1 5 10 15Ala Ala Pro Val Lys Gly 203514PRTArtificial Sequencesynthetic affinity ligand peptide sequence 35Met Gly Tyr Tyr Thr Ala Gly Gln Ala His Ala Tyr Asp Phe1 5 103617PRTArtificial Sequencesynthetic affinity ligand peptide sequence 36Asp Met Gly Thr Ser Tyr Leu Pro Ser Asn Trp Ser Tyr Pro Phe Ala1 5 10 15Tyr3711PRTArtificial Sequencesynthetic affinity ligand peptide sequence 37Ser Arg Ala Ser Tyr Ser Tyr Gly Phe Asp Tyr1 5 103815PRTArtificial Sequencesynthetic affinity ligand peptide sequence 38Ser Gln Arg Gly Gly Ala Ser Val Tyr Ser Tyr Ala Phe Asp Ile1 5 10 153910PRTArtificial Sequencesynthetic affinity ligand peptide sequence 39Gly Arg Gly Tyr Ser Tyr Pro Phe Asp Tyr1 5 104011PRTArtificial Sequencesynthetic affinity ligand peptide sequence 40Asp Ser Tyr Thr Ser Thr Gly Gly Met Asp Ile1 5 104112PRTArtificial Sequencesynthetic affinity ligand peptide sequence 41Asp His Ser Tyr Tyr Pro Val Phe Tyr Phe Asp Asn1 5 104210PRTArtificial Sequencesynthetic affinity ligand peptide sequence 42Ser Glu Tyr Ala Ile Val Tyr Phe Asp Tyr1 5 104313PRTArtificial Sequencesynthetic affinity ligand peptide sequence 43Ser Arg Thr Leu Val Ser Gly Tyr Tyr Pro Phe Asp Val1 5 104412PRTArtificial Sequencesynthetic affinity ligand peptide sequence 44Met Gly Tyr Tyr Pro Pro Ala Gly Ala Met Asp Val1 5 104511PRTArtificial Sequencesynthetic affinity ligand peptide sequence 45Val Arg Gly Tyr Tyr Ser Tyr Pro Phe Asp Val1 5 104613PRTArtificial Sequencesynthetic affinity ligand peptide sequence 46Ser Ile Lys Thr Tyr Tyr Val Tyr Gln Ala Phe Asp His1 5 104715PRTArtificial Sequencesynthetic affinity ligand peptide sequence 47Thr Arg Arg Gly Thr Trp Tyr Arg Tyr Ala Arg Ser Leu Asp Val1 5 10 154811PRTArtificial Sequencesynthetic affinity ligand peptide sequence 48Tyr Ala Tyr Ala Ala Gly Thr Ile Phe Asp Val1 5 104911PRTArtificial Sequencesynthetic affinity ligand peptide sequence 49Tyr Ala Tyr Ala Ala Gly Thr Ile Phe Asp Val1 5 105010PRTArtificial Sequencesynthetic affinity ligand peptide sequence 50Glu Met Gly Tyr Tyr Gln Gly Phe Asp Ile1 5 10516PRTArtificial Sequencesynthetic affinity ligand peptide sequence 51Glu Ser Tyr Phe Asp Tyr1 55211PRTArtificial Sequencesynthetic affinity ligand peptide sequence 52Ser Gly Asp Lys Ile Gly Lys Lys Tyr Ala Tyr1 5 105311PRTArtificial Sequencesynthetic affinity ligand peptide sequence 53Ser Gly Asp Asn Ile Gly Ser Lys Phe Ala Ser1 5 105411PRTArtificial Sequencesynthetic affinity ligand peptide sequence 54Ser Gly Asp Asn Leu Gly Asp Lys Phe Ala His1 5 105511PRTArtificial Sequencesynthetic affinity ligand peptide sequence 55Ser Gly Asp Ala Leu Gly Thr Gln Phe Ala His1 5 105611PRTArtificial Sequencesynthetic affinity ligand peptide sequence 56Ser Gly Asp Ala Leu Pro Thr Met Phe Ala His1 5 105711PRTArtificial Sequencesynthetic affinity ligand peptide sequence 57Ser Gly Asp Ser Leu Val Lys Lys His Ala Ser1 5 105811PRTArtificial Sequencesynthetic affinity ligand peptide sequence 58Ser Gly Asp Asn Leu Gly Lys Lys Tyr Val His1 5 105911PRTArtificial Sequencesynthetic affinity ligand peptide sequence 59Arg Ala Ser Gln Gly Ile Ser Ser Tyr Leu Asn1 5 106011PRTArtificial Sequencesynthetic affinity ligand peptide sequence 60Ser Gly Asp Asn Leu Gly Phe Lys Phe Ala His1 5 106111PRTArtificial Sequencesynthetic affinity ligand peptide sequence 61Ser Gly Asp Asn Ile Arg Thr Gln Phe Val Gln1 5 106211PRTArtificial Sequencesynthetic affinity ligand peptide sequence 62Ser Gly Asp Ala Ile Gly Asp Lys Phe Val His1 5 106311PRTArtificial Sequencesynthetic affinity ligand peptide sequence 63Ser Gly Asp Ala Leu Gly Thr Lys Tyr Val His1 5 106413PRTArtificial Sequencesynthetic affinity ligand peptide sequence 64Ser Gly Ser Ser Ser Asn Ile Gly Tyr Asn Tyr Val Ser1 5 106513PRTArtificial Sequencesynthetic affinity ligand peptide sequence 65Ser Gly Ser Ser Ser Asn Ile Gly Ala Asn Thr Val Ser1 5 106613PRTArtificial Sequencesynthetic affinity ligand peptide sequence 66Ser Gly Ser Ser Ser Asn Ile Gly Ala Asn Thr Val Ser1 5 106711PRTArtificial Sequencesynthetic affinity ligand peptide sequence 67Arg Ala Ser Gln Ser Ile Ile Asn Tyr Leu Asn1 5 106811PRTArtificial Sequencesynthetic affinity ligand peptide sequence 68Ser Gly Asp Asn Leu Gly Glu Lys Phe Val His1 5 106911PRTArtificial Sequencesynthetic affinity ligand peptide sequence 69Leu Val Ile Tyr Ser Asp Asn Asn Arg Pro Ser1 5 107011PRTArtificial Sequencesynthetic affinity ligand peptide sequence 70Leu Val Ile Tyr Asp Asp Ser Lys Arg Pro Ser1 5 107111PRTArtificial Sequencesynthetic affinity ligand peptide sequence 71Leu Val Ile Tyr Asp Asp Asn Asp Arg Pro Ser1 5 107211PRTArtificial Sequencesynthetic affinity ligand peptide sequence 72Leu Val Ile Tyr Asp Asp Asn Lys Arg Pro Ser1 5 107311PRTArtificial Sequencesynthetic affinity ligand peptide sequence 73Leu Val Ile Tyr Asp Asp Asn Lys Arg Pro Ser1 5 107411PRTArtificial Sequencesynthetic affinity ligand peptide sequence 74Leu Val Ile Tyr Asp Asp Asp Lys Arg Pro Ser1 5 107511PRTArtificial Sequencesynthetic affinity ligand peptide sequence 75Pro Val Ile Tyr Asp Asp Ser Lys Arg Pro Ser1 5 107611PRTArtificial Sequencesynthetic affinity ligand peptide sequence 76Leu Leu Ile Tyr Ser Ala Ser Asn Leu Gln Ser1 5 107711PRTArtificial Sequencesynthetic affinity ligand peptide sequence 77Leu Val Ile Tyr Asp Asp Ser Asn Arg Pro Ser1 5 107811PRTArtificial Sequencesynthetic affinity ligand peptide sequence 78Leu Val Ile Tyr Asp Asp Asn His Arg Pro Ser1 5 107911PRTArtificial Sequencesynthetic affinity ligand peptide sequence 79Leu Val Ile Tyr Asp Asp Ser Lys Arg Pro Ser1 5 108011PRTArtificial Sequencesynthetic affinity ligand peptide sequence 80Leu Val Ile Ser Asp Asp Asn Glu Arg Pro Ser1 5 108111PRTArtificial Sequencesynthetic affinity ligand peptide sequence 81Leu Leu Ile Tyr Ser Asn Thr Lys Arg Pro Ser1 5 108211PRTArtificial Sequencesynthetic affinity ligand peptide sequence 82Leu Leu Ile Tyr Gly Asn Ile Gln Arg Pro Ser1 5 108311PRTArtificial Sequencesynthetic affinity ligand peptide sequence 83Leu Leu Ile Tyr Gly Asn Ile Gln His Pro Ser1 5 108411PRTArtificial Sequencesynthetic affinity ligand peptide sequence 84Leu Leu Ile Ser Asp Ala Ser Ser Leu Gln Ser1 5 108511PRTArtificial Sequencesynthetic affinity ligand peptide sequence 85Leu Val Ile Tyr Tyr Asp Asn His Arg Pro Ser1 5 108610PRTArtificial Sequencesynthetic affinity ligand peptide sequence 86Tyr Val Thr Asp Gly Tyr Phe Thr Thr Gly1 5 108710PRTArtificial Sequencesynthetic affinity ligand peptide sequence 87Tyr Ser Arg Ala Gln Ser Gly Ser Pro Val1 5 108810PRTArtificial Sequencesynthetic affinity ligand peptide sequence 88Gln Ser Tyr Asp Ser Ser Ser Ser Leu Arg1 5 10898PRTArtificial Sequencesynthetic affinity ligand peptide sequence 89Gln Ser Ala Asp Trp Met Asp Tyr1 59010PRTArtificial Sequencesynthetic affinity ligand peptide sequence 90Ala Ser Tyr Ala Ser Ser Leu Asn Pro Val1 5 109110PRTArtificial Sequencesynthetic affinity ligand peptide sequence 91Ala Ser Tyr Asp Gly Trp Gly Asn Glu Arg1 5 109210PRTArtificial Sequencesynthetic affinity ligand peptide sequence 92Gln Ser Tyr Asp Arg Ser Leu Asp Phe Asn1 5 10938PRTArtificial Sequencesynthetic affinity ligand peptide sequence 93Gln Gln Gly Ile Ser Trp Leu Arg1 5948PRTArtificial Sequencesynthetic affinity ligand peptide sequence 94Ser Ser Tyr Asp Tyr Ser Ser Val1 59510PRTArtificial Sequencesynthetic affinity ligand peptide sequence 95Ala Ser Arg Asp Lys Ser Ala Asn Ser Val1 5 109610PRTArtificial Sequencesynthetic affinity ligand peptide sequence 96Gln Ser Tyr Asp Phe Gly Gly Asn Gly Ile1 5 109710PRTArtificial Sequencesynthetic affinity ligand peptide sequence 97Gln Ser Tyr Asp Phe Ser Ala Ser Ser Val1 5 109810PRTArtificial Sequencesynthetic affinity ligand peptide sequence 98Gln Ser Arg Ala His Gly Gly Asn Ser Ile1 5 109910PRTArtificial Sequencesynthetic affinity ligand peptide sequence 99Ala Ala Tyr Asp Ala Ile Phe Asn Lys Ile1 5 1010010PRTArtificial Sequencesynthetic affinity ligand peptide sequence 100Ala Ala Tyr Asp Ala Ile Phe Asn Lys Ile1 5 101018PRTArtificial Sequencesynthetic affinity ligand peptide sequence 101Gln Gln Asn Leu Ser Gly Pro Phe1 51028PRTArtificial Sequencesynthetic affinity ligand peptide sequence 102Ala Ser Trp Asp Ile Glu Ser Val1 510310PRTArtificial Sequencesynthetic affinity ligand peptide sequence 103Gly Gly Thr Phe Ser Ser Tyr Ser Ile Ser1 5 1010410PRTArtificial Sequencesynthetic affinity ligand peptide sequence 104Gly Gly Thr Phe Arg Ser Tyr Ala Ile Ser1 5 1010510PRTArtificial Sequencesynthetic affinity ligand peptide sequence 105Gly Gly Thr Phe Ser Ser Tyr Ala Ile Ser1 5 1010610PRTArtificial Sequencesynthetic affinity ligand peptide sequence 106Gly Gly Thr Phe Ser Ser Tyr Ser Ile Ser1 5 1010710PRTArtificial Sequencesynthetic affinity ligand peptide sequence 107Gly Gly Thr Phe Ser Ser Asn Thr Ile Ser1 5 1010810PRTArtificial Sequencesynthetic affinity ligand peptide sequence 108Gly Gly Thr Phe Ser Ser Tyr Ala Ile Ser1 5 1010910PRTArtificial Sequencesynthetic affinity ligand peptide sequence 109Gly Tyr Thr Phe Thr Ser Tyr Tyr Ile His1 5 1011010PRTArtificial Sequencesynthetic affinity ligand peptide sequence 110Gly Gly Thr Phe Ser Ser Tyr Ala Ile Asn1 5 1011110PRTArtificial Sequencesynthetic affinity ligand peptide sequence 111Gly Gly Thr Phe Ser Ser Tyr Ala Ile Ser1

5 1011210PRTArtificial Sequencesynthetic affinity ligand peptide sequence 112Gly Gly Thr Phe Ser Ser Tyr Ala Ile Gly1 5 1011310PRTArtificial Sequencesynthetic affinity ligand peptide sequence 113Gly Gly Thr Phe Ser Ser Tyr Ala Ile Ser1 5 1011410PRTArtificial Sequencesynthetic affinity ligand peptide sequence 114Gly Gly Thr Phe Ser Ser Tyr Ala Ile Asn1 5 1011510PRTArtificial Sequencesynthetic affinity ligand peptide sequence 115Gly Gly Thr Phe Ser Gly Tyr Gly Ile Ser1 5 1011610PRTArtificial Sequencesynthetic affinity ligand peptide sequence 116Gly Phe Thr Phe Ser Asp Tyr Gly Leu His1 5 1011710PRTArtificial Sequencesynthetic affinity ligand peptide sequence 117Gly Gly Thr Phe Ser Ser Tyr Ala Val Asn1 5 1011810PRTArtificial Sequencesynthetic affinity ligand peptide sequence 118Gly Gly Thr Phe Ser Ser Tyr Ala Ile Ser1 5 1011910PRTArtificial Sequencesynthetic affinity ligand peptide sequence 119Gly Gly Thr Phe Ser Thr Tyr Ala Ile Ser1 5 1012010PRTArtificial Sequencesynthetic affinity ligand peptide sequence 120Gly Gly Thr Phe Arg Ser Tyr Ala Val His1 5 1012110PRTArtificial Sequencesynthetic affinity ligand peptide sequence 121Gly Gly Thr Phe Ser Asp Asn Ala Ile Ser1 5 1012210PRTArtificial Sequencesynthetic affinity ligand peptide sequence 122Gly Gly Thr Phe Ser Ser Tyr Ala Ile Ser1 5 1012320PRTArtificial Sequencesynthetic affinity ligand peptide sequence 123Trp Met Gly Gly Ile Ile Pro Ile Phe Gly Ile Ala Ser Tyr Ala Gln1 5 10 15Lys Phe Gln Gly 2012420PRTArtificial Sequencesynthetic affinity ligand peptide sequence 124Trp Met Gly Gly Ile Ile Pro Arg Phe Gly Ile Ala Asn Tyr Ala Gln1 5 10 15Lys Phe Gln Gly 2012520PRTArtificial Sequencesynthetic affinity ligand peptide sequence 125Trp Met Gly Gly Ile Tyr Pro Phe Val Gly Thr Ala His Tyr Ala Gln1 5 10 15Lys Phe Gln Gly 2012620PRTArtificial Sequencesynthetic affinity ligand peptide sequence 126Trp Met Gly Gly Ile Ile Pro Ile Phe Gly Ser Ala Asn Tyr Ala Gln1 5 10 15Lys Phe Gln Gly 2012720PRTArtificial Sequencesynthetic affinity ligand peptide sequence 127Trp Met Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln1 5 10 15Lys Phe Gln Gly 2012820PRTArtificial Sequencesynthetic affinity ligand peptide sequence 128Trp Met Gly Gly Ile Ile Pro Ile Phe Gly Ile Ala Lys Tyr Ala Gln1 5 10 15Lys Phe Gln Gly 2012920PRTArtificial Sequencesynthetic affinity ligand peptide sequence 129Trp Met Gly Trp Ile Asn Pro Asn Asn Gly Asn Thr Arg Tyr Ala Gln1 5 10 15Lys Phe Gln Gly 2013020PRTArtificial Sequencesynthetic affinity ligand peptide sequence 130Trp Met Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln1 5 10 15Lys Phe Gln Gly 2013120PRTArtificial Sequencesynthetic affinity ligand peptide sequence 131Trp Met Gly Gly Ile Ile Pro Ile Phe Gly Ile Ala Lys Tyr Ala Gln1 5 10 15Lys Phe Gln Gly 2013220PRTArtificial Sequencesynthetic affinity ligand peptide sequence 132Trp Met Gly Gly Ile Ile Pro His Phe Gly Thr Ala Asn Tyr Ala Gln1 5 10 15Lys Phe Gln Gly 2013320PRTArtificial Sequencesynthetic affinity ligand peptide sequence 133Trp Met Gly Gly Ile His Pro Ala Phe Gly Thr Ala Thr Tyr Ala Gln1 5 10 15Lys Phe Gln Gly 2013420PRTArtificial Sequencesynthetic affinity ligand peptide sequence 134Trp Met Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln1 5 10 15Lys Phe Gln Gly 2013520PRTArtificial Sequencesynthetic affinity ligand peptide sequence 135Trp Met Gly Gly Ile Tyr Pro Ile Phe Gly Tyr Ala Asn Tyr Ala Gln1 5 10 15Lys Phe Gln Gly 2013622PRTArtificial Sequencesynthetic affinity ligand peptide sequence 136Trp Val Gly Arg Ile Lys Ser Lys Thr Asn Gly Gly Thr Thr Asp Tyr1 5 10 15Ala Ala Pro Val Lys Gly 2013720PRTArtificial Sequencesynthetic affinity ligand peptide sequence 137Trp Met Gly Gly Ile Ala Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln1 5 10 15Lys Phe Gln Gly 2013820PRTArtificial Sequencesynthetic affinity ligand peptide sequence 138Trp Met Gly Gly Ile Ile Pro His Phe Gly Thr Ala Ser Tyr Ala Gln1 5 10 15Lys Phe Gln Gly 2013920PRTArtificial Sequencesynthetic affinity ligand peptide sequence 139Trp Met Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln1 5 10 15Lys Phe Gln Gly 2014020PRTArtificial Sequencesynthetic affinity ligand peptide sequence 140Trp Met Gly Gly Ile Ile Pro Asn Phe Gly Thr Ala His Tyr Ala Gln1 5 10 15Lys Phe Gln Gly 2014120PRTArtificial Sequencesynthetic affinity ligand peptide sequence 141Trp Met Gly Gly Ile Ile Pro His Phe Gly Thr Ala Asn Tyr Ala Gln1 5 10 15Lys Phe Gln Gly 2014220PRTArtificial Sequencesynthetic affinity ligand peptide sequence 142Trp Met Gly Gly Ile Ile Pro Ile Phe Gly Ala Ala Thr Tyr Ala Gln1 5 10 15Lys Phe Gln Gly 2014310PRTArtificial Sequencesynthetic affinity ligand peptide sequence 143Asp Ser Ser Ser Ile Glu Tyr Phe Asp Val1 5 1014410PRTArtificial Sequencesynthetic affinity ligand peptide sequence 144Gly His Arg Tyr Thr Asp Gly Phe Ala Tyr1 5 1014510PRTArtificial Sequencesynthetic affinity ligand peptide sequence 145Asp Arg Ser Ile Tyr Ser Tyr Phe Asp Tyr1 5 101469PRTArtificial Sequencesynthetic affinity ligand peptide sequence 146His Gly Val Ser Glu Ala Phe Asp Tyr1 514710PRTArtificial Sequencesynthetic affinity ligand peptide sequence 147Glu Val Asp Ser Ser Tyr Pro Glu Asp Tyr1 5 1014810PRTArtificial Sequencesynthetic affinity ligand peptide sequence 148Asp Ile Arg Ile Ser Thr His Phe Asp Tyr1 5 1014910PRTArtificial Sequencesynthetic affinity ligand peptide sequence 149Asn Ser Phe Tyr Ser Glu Trp Phe Asp Val1 5 1015017PRTArtificial Sequencesynthetic affinity ligand peptide sequence 150Val Leu Tyr Ser Ser Tyr Tyr Gly Met Gly His Tyr Glu Tyr Phe Asp1 5 10 15Ile15110PRTArtificial Sequencesynthetic affinity ligand peptide sequence 151Asp Ile Arg Ile Ser Thr His Phe Asp Tyr1 5 1015216PRTArtificial Sequencesynthetic affinity ligand peptide sequence 152His Ser Tyr Ser Thr Gly Leu Tyr Met Gly Ser Asp Tyr Met Asp Tyr1 5 10 1515314PRTArtificial Sequencesynthetic affinity ligand peptide sequence 153His Ala Gly Tyr Gly Ala Ser Gly Tyr Glu Tyr Met Asp Asn1 5 1015419PRTArtificial Sequencesynthetic affinity ligand peptide sequence 154Asp Val Ser Ser Tyr Tyr Tyr Tyr Gly Phe His Tyr His Ala Tyr Trp1 5 10 15Phe Asp Val1557PRTArtificial Sequencesynthetic affinity ligand peptide sequence 155Asp Ser Asp Tyr Phe Asp Tyr1 515613PRTArtificial Sequencesynthetic affinity ligand peptide sequence 156Ser Lys Gly Arg Gly Leu Tyr Gln Asn Ile Gln Asp Tyr1 5 1015711PRTArtificial Sequencesynthetic affinity ligand peptide sequence 157Gly His Tyr Ile Ser Ser Tyr Ala Phe Asp Val1 5 1015810PRTArtificial Sequencesynthetic affinity ligand peptide sequence 158Asp Ile Arg Ile Ser Thr His Phe Asp Tyr1 5 101598PRTArtificial Sequencesynthetic affinity ligand peptide sequence 159Asp Phe Tyr Asp Gly Phe Asp Tyr1 516010PRTArtificial Sequencesynthetic affinity ligand peptide sequence 160Asp Glu Tyr Val Gly His Tyr Phe Asp His1 5 1016111PRTArtificial Sequencesynthetic affinity ligand peptide sequence 161Glu Pro Ile Val Asn Ser Ser Pro Met Ala Val1 5 1016210PRTArtificial Sequencesynthetic affinity ligand peptide sequence 162Gln Glu Tyr Ser Tyr Tyr Asn Phe Asp Pro1 5 1016311PRTArtificial Sequencesynthetic affinity ligand peptide sequence 163Arg Ala Ser Gln Ser Ile Asn Thr Tyr Leu Ala1 5 1016411PRTArtificial Sequencesynthetic affinity ligand peptide sequence 164Arg Ala Ser Gln Gly Ile Ser Asn His Leu Asn1 5 1016513PRTArtificial Sequencesynthetic affinity ligand peptide sequence 165Ser Gly Ser Ser Ser Asn Ile Gly Lys Asn Tyr Val His1 5 1016611PRTArtificial Sequencesynthetic affinity ligand peptide sequence 166Arg Ala Ser Gln Ser Ile Ser Asn Tyr Leu Asn1 5 1016711PRTArtificial Sequencesynthetic affinity ligand peptide sequence 167Arg Ala Ser Gln Asp Ile Met Leu Asn Leu Asn1 5 1016811PRTArtificial Sequencesynthetic affinity ligand peptide sequence 168Arg Ala Ser Gln Ser Ile Arg Asn Tyr Leu Ala1 5 1016911PRTArtificial Sequencesynthetic affinity ligand peptide sequence 169Arg Ala Ser Gln Ser Ile Asn Ser Tyr Leu Ala1 5 1017012PRTArtificial Sequencesynthetic affinity ligand peptide sequence 170Arg Ala Ser Gln Ile Val Ser Ser Ser Tyr Leu Val1 5 1017111PRTArtificial Sequencesynthetic affinity ligand peptide sequence 171Arg Ala Ser Gln Gly Ile Leu Ser Phe Leu Thr1 5 1017211PRTArtificial Sequencesynthetic affinity ligand peptide sequence 172Arg Ala Ser Gln Asp Ile Ser Arg Tyr Leu Asn1 5 1017311PRTArtificial Sequencesynthetic affinity ligand peptide sequence 173Arg Ala Ser Gln Ser Ile Ile Lys Tyr Leu Ala1 5 1017411PRTArtificial Sequencesynthetic affinity ligand peptide sequence 174Ser Gly Asp Asn Ile Arg Lys Lys Tyr Val His1 5 1017511PRTArtificial Sequencesynthetic affinity ligand peptide sequence 175Arg Ala Ser Gln Asp Ile Arg Asn Tyr Leu Asn1 5 1017611PRTArtificial Sequencesynthetic affinity ligand peptide sequence 176Ser Gly Asp Lys Ile Gly Asp Lys Tyr Ala Asp1 5 1017711PRTArtificial Sequencesynthetic affinity ligand peptide sequence 177Ser Gly Asp Lys Leu Gly Ser Ser Tyr Ala Thr1 5 1017811PRTArtificial Sequencesynthetic affinity ligand peptide sequence 178Arg Ala Ser Gln Ser Ile Arg Asn Tyr Leu Ala1 5 1017911PRTArtificial Sequencesynthetic affinity ligand peptide sequence 179Arg Ala Ser Gln Ser Ile Lys Thr Tyr Leu Ala1 5 1018011PRTArtificial Sequencesynthetic affinity ligand peptide sequence 180Arg Ala Ser Gln Ser Ile Phe Asn Tyr Leu Asn1 5 1018111PRTArtificial Sequencesynthetic affinity ligand peptide sequence 181Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn1 5 1018211PRTArtificial Sequencesynthetic affinity ligand peptide sequence 182Arg Ala Ser Gln Ser Ile Asn Asn Tyr Leu Asn1 5 1018311PRTArtificial Sequencesynthetic affinity ligand peptide sequence 183Leu Leu Ile Ser Gly Ala Ser Ser Leu Gln Ser1 5 1018411PRTArtificial Sequencesynthetic affinity ligand peptide sequence 184Leu Leu Ile Tyr Gly Ala Ser Ser Leu Gln Ser1 5 1018511PRTArtificial Sequencesynthetic affinity ligand peptide sequence 185Val Leu Ile Tyr Arg Asp Asn Gln Arg Pro Ser1 5 1018611PRTArtificial Sequencesynthetic affinity ligand peptide sequence 186Leu Leu Ile Tyr Asp Ala Ser Ser Leu Gln Ser1 5 1018711PRTArtificial Sequencesynthetic affinity ligand peptide sequence 187Leu Leu Ile Tyr Ala Thr Ser Ser Leu Gln Ser1 5 1018811PRTArtificial Sequencesynthetic affinity ligand peptide sequence 188Leu Leu Ile Tyr Asp Ala Ser Ser Leu Gln Ser1 5 1018911PRTArtificial Sequencesynthetic affinity ligand peptide sequence 189Leu Leu Ile Tyr Ala Ala Ser Asn Leu Gln Ser1 5 1019011PRTArtificial Sequencesynthetic affinity ligand peptide sequence 190Leu Leu Ile Tyr Gly Ala Ser Ser Arg Ala Thr1 5 1019111PRTArtificial Sequencesynthetic affinity ligand peptide sequence 191Leu Leu Ile Tyr Asp Ala Ser Ser Leu Gln Ser1 5 1019211PRTArtificial Sequencesynthetic affinity ligand peptide sequence 192Leu Leu Ile Tyr Gly Ala Ser Asn Leu Gln Ser1 5 1019311PRTArtificial Sequencesynthetic affinity ligand peptide sequence 193Leu Leu Ile Tyr Gly Ala Ser Lys Leu Gln Ser1 5 1019411PRTArtificial Sequencesynthetic affinity ligand peptide sequence 194Leu Val Ile Tyr Asp Asp Asn Glu Arg Pro Ser1 5 1019511PRTArtificial Sequencesynthetic affinity ligand peptide sequence 195Leu Leu Ile Tyr Gln Val Ser Thr Gln Gln Ser1 5 1019611PRTArtificial Sequencesynthetic affinity ligand peptide sequence 196Leu Val Ile Tyr Arg Asp Ser Asn Arg Pro Ser1 5 1019711PRTArtificial Sequencesynthetic affinity ligand peptide sequence 197Leu Val Ile Tyr Glu Gln Ser Lys Arg Pro Ser1 5 1019811PRTArtificial Sequencesynthetic affinity ligand peptide sequence 198Leu Leu Ile Tyr Asp Ala Ser Ser Leu Gln Ser1 5 1019911PRTArtificial Sequencesynthetic affinity ligand peptide sequence 199Leu Leu Ile Tyr Ala Val Ser Ser Leu Gln Ser1 5 1020011PRTArtificial Sequencesynthetic affinity ligand peptide sequence 200Leu Leu Ile Tyr Ala Ala Ser Arg Leu Gln Ser1 5 1020111PRTArtificial Sequencesynthetic affinity ligand peptide sequence 201Leu Leu Ile His Asp Ala Ser Ser Leu Gln Ser1 5 1020211PRTArtificial Sequencesynthetic affinity ligand peptide sequence 202Leu Leu Ile Tyr Gln Ala Ser Arg Leu Gln Ser1 5 102038PRTArtificial Sequencesynthetic affinity ligand peptide sequence 203Gln Gln Ala Tyr Thr Arg Ser Phe1 52048PRTArtificial Sequencesynthetic affinity ligand peptide sequence 204Gln Gln Glu Tyr Ser Ser Pro Ile1 520510PRTArtificial Sequencesynthetic affinity ligand peptide sequence 205Gln Ala Tyr Asp Leu Leu Ser Arg Arg Trp1 5 102068PRTArtificial Sequencesynthetic affinity ligand peptide sequence 206Gln Gln Tyr Tyr His Phe Pro Ile1 52078PRTArtificial Sequencesynthetic affinity ligand peptide sequence 207Gln Gln Arg Ser His Trp Ser Asn1 52088PRTArtificial Sequencesynthetic affinity ligand peptide sequence 208His Gln Tyr Tyr Ser Thr Pro Leu1 52098PRTArtificial Sequencesynthetic affinity ligand peptide sequence 209Gln Gln Tyr Tyr Ser Trp Pro Ile1 52108PRTArtificial Sequencesynthetic affinity ligand peptide sequence 210Gln Gln Ala Asp Gln Tyr Pro Met1 52118PRTArtificial Sequencesynthetic affinity ligand peptide sequence 211His Gln Tyr Tyr Ser Thr Pro Leu1 52128PRTArtificial Sequencesynthetic affinity ligand peptide sequence 212Gln Gln Ala Tyr Ser Thr Pro Val1 52138PRTArtificial Sequencesynthetic affinity ligand peptide sequence 213Gln Gln Tyr Tyr Ser Tyr Pro Ala1 52148PRTArtificial Sequencesynthetic affinity ligand peptide sequence 214Gln Val Ala Thr Tyr Leu Asn Arg1 52158PRTArtificial Sequencesynthetic affinity ligand peptide sequence 215Gln Gln Ala Tyr Ser Asn Pro His1 521610PRTArtificial Sequencesynthetic affinity ligand peptide sequence 216Ala Ser Tyr Asp Trp His Met Ile His Tyr1 5 102178PRTArtificial Sequencesynthetic affinity ligand peptide sequence 217Gln Val Trp Thr Arg Thr Gln Tyr1 52188PRTArtificial Sequencesynthetic affinity ligand peptide sequence 218His Gln Tyr Tyr Ser Thr Pro Leu1 52198PRTArtificial Sequencesynthetic affinity ligand peptide sequence 219Met Gln Ser Tyr Ser Ser Pro Tyr1 52208PRTArtificial Sequencesynthetic affinity ligand peptide sequence 220Gln Gln Met Tyr Asp Lys Pro Phe1 52217PRTArtificial Sequencesynthetic affinity ligand peptide sequence

221Gln Gln Ser Leu Gln Tyr Tyr1 52228PRTArtificial Sequencesynthetic affinity ligand peptide sequence 222Gln Gln Gly Tyr Ser Ser Pro Phe1 522312PRTArtificial Sequencesynthetic affinity ligand peptide sequence 223Gly Asp Ser Val Ser Asp Ser Ser Ala Ala Trp Asn1 5 1022410PRTArtificial Sequencesynthetic affinity ligand peptide sequence 224Gly Phe Thr Phe Ser Arg Tyr Gly Met Asn1 5 1022510PRTArtificial Sequencesynthetic affinity ligand peptide sequence 225Gly Phe Thr Phe Gly Asp Tyr Trp Ile His1 5 1022610PRTArtificial Sequencesynthetic affinity ligand peptide sequence 226Gly Phe Thr Phe Ser Arg Tyr Ala Met Ser1 5 1022710PRTArtificial Sequencesynthetic affinity ligand peptide sequence 227Gly Gly Thr Phe Ser Gly Tyr Ala Ile Ser1 5 1022810PRTArtificial Sequencesynthetic affinity ligand peptide sequence 228Gly Tyr Ser Phe Thr Thr Tyr Thr Ile Ser1 5 1022910PRTArtificial Sequencesynthetic affinity ligand peptide sequence 229Gly Phe Thr Phe Ser Ser Tyr Gly Met His1 5 1023010PRTArtificial Sequencesynthetic affinity ligand peptide sequence 230Gly Phe Thr Phe Ser Ser Phe Ala Leu Thr1 5 1023121PRTArtificial Sequencesynthetic affinity ligand peptide sequence 231Trp Leu Gly Arg Ile Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala1 5 10 15Val Ser Val Lys Ser 2023220PRTArtificial Sequencesynthetic affinity ligand peptide sequence 232Trp Val Ser Gly Ile Ser Gly Ser Gly Ser Tyr Thr Tyr Tyr Ala Asp1 5 10 15Ser Val Lys Gly 2023319PRTArtificial Sequencesynthetic affinity ligand peptide sequence 233Trp Val Ser Ser Ile Ser Gly Gly Gly Asn Thr Tyr Tyr Ala Asp Ser1 5 10 15Val Lys Gly23420PRTArtificial Sequencesynthetic affinity ligand peptide sequence 234Trp Val Ser Ser Ile Ser Tyr Lys Gly Ser Asn Thr Tyr Tyr Ala Asp1 5 10 15Ser Val Lys Gly 2023520PRTArtificial Sequencesynthetic affinity ligand peptide sequence 235Trp Met Gly Arg Ile Phe Pro Arg Ser Gly Phe Ala Asn Tyr Ala Gln1 5 10 15Lys Phe Gln Gly 2023620PRTArtificial Sequencesynthetic affinity ligand peptide sequence 236Trp Met Gly Ile Ile Tyr Pro Ser Asp Ser Asp Thr Ile Tyr Ser Pro1 5 10 15Ser Phe Gln Gly 2023722PRTArtificial Sequencesynthetic affinity ligand peptide sequence 237Trp Val Gly Arg Ile Lys Ser Lys Met Asn Gly Gly Thr Thr Asp Tyr1 5 10 15Ala Ala Pro Val Lys Gly 2023822PRTArtificial Sequencesynthetic affinity ligand peptide sequence 238Trp Val Gly Phe Ile Lys Ser Lys Thr His Gly Gly Thr Thr Asp Tyr1 5 10 15Ala Ala Pro Val Lys Gly 2023913PRTArtificial Sequencesynthetic affinity ligand peptide sequence 239Glu Ser Pro Ala Asp Val Ser Gly Ile Asn Phe Asp Ile1 5 1024012PRTArtificial Sequencesynthetic affinity ligand peptide sequence 240Arg Ser Arg Tyr Pro Tyr Val Tyr Val Phe Asp Tyr1 5 1024112PRTArtificial Sequencesynthetic affinity ligand peptide sequence 241Ser Leu Tyr Trp Arg Tyr Ser Ser Tyr Phe Asp Pro1 5 1024215PRTArtificial Sequencesynthetic affinity ligand peptide sequence 242Ala Pro Tyr Pro Gly Ser Val Ser Arg Tyr Gly Ala Phe Asp Tyr1 5 10 1524314PRTArtificial Sequencesynthetic affinity ligand peptide sequence 243Asp Val Ser Gly Val Thr Gly Tyr Arg Lys Ala Arg Asp Tyr1 5 102448PRTArtificial Sequencesynthetic affinity ligand peptide sequence 244Ser Ser Val Val Gly Phe Asp Val1 52458PRTArtificial Sequencesynthetic affinity ligand peptide sequence 245Ser Leu Thr Ser Gly Phe Asp Tyr1 52467PRTArtificial Sequencesynthetic affinity ligand peptide sequence 246Asn Arg Gly His Phe Asp Tyr1 524711PRTArtificial Sequencesynthetic affinity ligand peptide sequence 247Arg Ala Ser Gln Ser Ile Tyr Ser His Leu Ala1 5 1024813PRTArtificial Sequencesynthetic affinity ligand peptide sequence 248Ser Gly Ser Ser Ser Asn Ile Gly Ser Tyr Tyr Val Asn1 5 1024911PRTArtificial Sequencesynthetic affinity ligand peptide sequence 249Arg Ala Ser Gln Thr Ile Ser Asn His Leu Asn1 5 1025014PRTArtificial Sequencesynthetic affinity ligand peptide sequence 250Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly Tyr Tyr Val His1 5 1025111PRTArtificial Sequencesynthetic affinity ligand peptide sequence 251Arg Ala Ser Gln Gly Ile Arg Thr Arg Leu Lys1 5 1025211PRTArtificial Sequencesynthetic affinity ligand peptide sequence 252Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn1 5 1025311PRTArtificial Sequencesynthetic affinity ligand peptide sequence 253Ser Gly Asp Asn Leu Arg Asp Lys Tyr Val Tyr1 5 1025413PRTArtificial Sequencesynthetic affinity ligand peptide sequence 254Ser Gly Ser Ser Ser Asn Ile Gly Ala Tyr Tyr Val Tyr1 5 1025511PRTArtificial Sequencesynthetic affinity ligand peptide sequence 255Leu Leu Ile Tyr Ala Ala Ser Asn Leu Gln Ser1 5 1025611PRTArtificial Sequencesynthetic affinity ligand peptide sequence 256Leu Leu Ile Tyr Arg Asn Asn Gln Arg Pro Ser1 5 1025711PRTArtificial Sequencesynthetic affinity ligand peptide sequence 257Leu Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser1 5 1025811PRTArtificial Sequencesynthetic affinity ligand peptide sequence 258Leu Leu Ile Tyr Gly Asn Asn Gln Arg Pro Ser1 5 1025911PRTArtificial Sequencesynthetic affinity ligand peptide sequence 259Leu Leu Ile Tyr Gly Ala Ser Thr Leu Gln Ser1 5 1026011PRTArtificial Sequencesynthetic affinity ligand peptide sequence 260Leu Leu Ile Tyr Ala Ala Ser Arg Leu Gln Ser1 5 1026111PRTArtificial Sequencesynthetic affinity ligand peptide sequence 261Leu Val Ile Tyr Ser Asn Ser Asn Arg Pro Ser1 5 1026211PRTArtificial Sequencesynthetic affinity ligand peptide sequence 262Leu Leu Ile Tyr Gly Asn Asn Gln Arg Pro Ser1 5 102637PRTArtificial Sequencesynthetic affinity ligand peptide sequence 263Gln Gln Ser Asp Glu Ser Ile1 526410PRTArtificial Sequencesynthetic affinity ligand peptide sequence 264Ser Ala Tyr Thr Gly Leu Ser Ser Ser Pro1 5 102658PRTArtificial Sequencesynthetic affinity ligand peptide sequence 265Gln Gln Ser Leu His Tyr Pro Tyr1 526610PRTArtificial Sequencesynthetic affinity ligand peptide sequence 266Gly Ala Arg Asp Phe Gln Leu Ser Ser Trp1 5 102678PRTArtificial Sequencesynthetic affinity ligand peptide sequence 267Gln Gln Gln Asp Gln Thr Pro Tyr1 52688PRTArtificial Sequencesynthetic affinity ligand peptide sequence 268Gln Gln His Leu Ser Trp Pro Glu1 526910PRTArtificial Sequencesynthetic affinity ligand peptide sequence 269Tyr Ala Trp Ala Arg Arg His Thr Gly Ala1 5 1027010PRTArtificial Sequencesynthetic affinity ligand peptide sequence 270Tyr Ser Trp Asp His Met Leu Asn Gly Tyr1 5 10271245PRTArtificial Sequencesynthetic affinity ligand peptide sequence 271Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr 20 25 30Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ser Ile Ser Tyr Lys Gly Ser Asn Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Ala Pro Tyr Pro Gly Ser Val Ser Arg Tyr Gly Ala Phe Asp 100 105 110Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys 115 120 125Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly 130 135 140Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro145 150 155 160Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 165 170 175Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 180 185 190Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn 195 200 205Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro 210 215 220Lys Ser Glu Phe Asp Tyr Lys Asp Asp Asp Asp Lys Gly Ala Pro His225 230 235 240His His His His His 245272216PRTArtificial Sequencesynthetic affinity ligand peptide sequence 272Asp Ile Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln1 5 10 15Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly 20 25 30Tyr Tyr Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45Leu Ile Tyr Gly Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu65 70 75 80Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Ala Arg Asp Phe Gln 85 90 95Leu Ser Ser Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu 115 120 125Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe 130 135 140Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val145 150 155 160Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys 165 170 175Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser 180 185 190His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu 195 200 205Lys Thr Val Ala Pro Thr Glu Ala 210 215273239PRTArtificial Sequencesynthetic affinity ligand peptide sequence 273Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30Ala Leu Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Phe Ile Lys Ser Lys Thr His Gly Gly Thr Thr Asp Tyr Ala Ala 50 55 60Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr65 70 75 80Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95Tyr Cys Ala Arg Asn Arg Gly His Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn145 150 155 160Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205Asn Thr Lys Val Asp Lys Lys Val Glu Thr Lys Ser Glu Phe Asp Tyr 210 215 220Lys Asp Asp Asp Asp Lys Gly Ala Pro His His His His His His225 230 235274215PRTArtificial Sequencesynthetic affinity ligand peptide sequence 274Asp Ile Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln1 5 10 15Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ala Tyr 20 25 30Tyr Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45Ile Tyr Gly Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu Gln65 70 75 80Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Tyr Ser Trp Asp His Met Leu 85 90 95Asn Gly Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln 100 105 110Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu 115 120 125Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr 130 135 140Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys145 150 155 160Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr 165 170 175Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His 180 185 190Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys 195 200 205Thr Val Ala Pro Thr Glu Ala 210 215275240PRTArtificial Sequencesynthetic affinity ligand peptide sequence 275Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Arg Ile Lys Ser Lys Met Asn Gly Gly Thr Thr Asp Tyr Ala Ala 50 55 60Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr65 70 75 80Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95Tyr Cys Ala Arg Ser Leu Thr Ser Gly Phe Asp Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp145 150 155 160Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Glu Phe Asp 210 215 220Tyr Lys Asp Asp Asp Asp Lys Gly Ala Pro His His His His His His225 230 235 240276213PRTArtificial Sequencesynthetic affinity ligand peptide sequence 276Asp Ile Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln1 5 10 15Thr Ala Ser Ile Thr Cys Ser Gly Asp Asn Leu Arg Asp Lys Tyr Val 20 25 30Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40 45Ser Asn Ser Asn Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Glu65 70 75 80Asp Glu Ala Asp Tyr Tyr Cys Tyr Ala Trp Ala Arg Arg His Thr Gly 85 90 95Ala Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys 100 105 110Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln 115 120 125Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly 130 135 140Ala Val Thr Val Ala Trp Lys

Ala Asp Ser Ser Pro Val Lys Ala Gly145 150 155 160Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala 165 170 175Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser 180 185 190Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val 195 200 205Ala Pro Thr Glu Ala 210277244PRTArtificial Sequencesynthetic affinity ligand peptide sequence 277Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Gly Tyr 20 25 30Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Arg Ile Phe Pro Arg Ser Gly Phe Ala Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp Val Ser Gly Val Thr Gly Tyr Arg Lys Ala Arg Asp Tyr 100 105 110Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 130 135 140Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val145 150 155 160Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170 175Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185 190Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 195 200 205Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 210 215 220Ser Glu Phe Asp Tyr Lys Asp Asp Asp Asp Lys Gly Ala Pro His His225 230 235 240His His His His278214PRTArtificial Sequencesynthetic affinity ligand peptide sequence 278Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Thr Arg 20 25 30Leu Lys Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Gly Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gln Asp Gln Thr Pro Tyr 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu Ala 210279241PRTArtificial Sequencesynthetic affinity ligand peptide sequence 279Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Gly Asp Tyr 20 25 30Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ser Ile Ser Gly Gly Gly Asn Thr Tyr Tyr Ala Asp Ser Val Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu65 70 75 80Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Arg Ser Leu Tyr Trp Arg Tyr Ser Ser Tyr Phe Asp Pro Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser145 150 155 160Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Glu Phe 210 215 220Asp Tyr Lys Asp Asp Asp Asp Lys Gly Ala Pro His His His His His225 230 235 240His280214PRTArtificial Sequencesynthetic affinity ligand peptide sequence 280Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Thr Ile Ser Asn His 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Leu His Tyr Pro Tyr 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu Ala 210281246PRTArtificial Sequencesynthetic affinity ligand peptide sequence 281Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1 5 10 15Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Asp Ser 20 25 30Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu 35 40 45Trp Leu Gly Arg Ile Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala 50 55 60Val Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn65 70 75 80Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val 85 90 95Tyr Tyr Cys Ala Arg Glu Ser Pro Ala Asp Val Ser Gly Ile Asn Phe 100 105 110Asp Ile Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr 115 120 125Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser 130 135 140Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu145 150 155 160Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 165 170 175Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 180 185 190Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys 195 200 205Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu 210 215 220Pro Lys Ser Glu Phe Asp Tyr Lys Asp Asp Asp Asp Lys Gly Ala Pro225 230 235 240His His His His His His 245282213PRTArtificial Sequencesynthetic affinity ligand peptide sequence 282Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Tyr Ser His 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala Ala Ser Asn Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Asp Glu Ser Ile Thr 85 90 95Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro 100 105 110Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr 115 120 125Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys 130 135 140Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu145 150 155 160Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser 165 170 175Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala 180 185 190Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe 195 200 205Asn Arg Gly Glu Ala 210283240PRTArtificial Sequencesynthetic affinity ligand peptide sequence 283Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr 20 25 30Asp Val Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ile Ile Asn Pro Tyr Asn Gly Lys Thr Lys Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Arg Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Lys Pro Val Gly Ala Arg Tyr Phe Asp Ile Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp145 150 155 160Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Glu Phe Asp 210 215 220Tyr Lys Asp Asp Asp Asp Lys Gly Ala Pro His His His His His His225 230 235 240284216PRTArtificial Sequencesynthetic affinity ligand peptide sequence 284Asp Ile Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln1 5 10 15Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly 20 25 30Tyr Val Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45Leu Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu65 70 75 80Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ser Trp Asp Leu Gly 85 90 95Arg Lys Tyr Ser Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu 115 120 125Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe 130 135 140Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val145 150 155 160Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys 165 170 175Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser 180 185 190His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu 195 200 205Lys Thr Val Ala Pro Thr Glu Ala 210 215285244PRTArtificial Sequencesynthetic affinity ligand peptide sequence 285Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Asp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Asn Pro Tyr Asn Gly Gly Thr Lys Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Arg Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Ala Ser Tyr Gly Gly Tyr Ser His Val Tyr Ser Phe Asp Ile 100 105 110Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 130 135 140Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val145 150 155 160Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170 175Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185 190Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 195 200 205Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 210 215 220Ser Glu Phe Asp Tyr Lys Asp Asp Asp Asp Lys Gly Ala Pro His His225 230 235 240His His His His286213PRTArtificial Sequencesynthetic affinity ligand peptide sequence 286Asp Ile Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln1 5 10 15Thr Ala Ser Ile Thr Cys Ser Gly Asp Lys Leu Arg Arg Lys Ile Val 20 25 30His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40 45Ser Asp Thr Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Glu65 70 75 80Asp Glu Ala Asp Tyr Tyr Cys Ala Ser Arg Thr Leu Gly Pro Arg Ile 85 90 95Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys 100 105 110Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln 115 120 125Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly 130 135 140Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly145 150 155 160Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala 165 170 175Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser 180 185 190Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val 195 200 205Ala Pro Thr Glu Ala 210287238PRTArtificial Sequencesynthetic affinity ligand peptide sequence 287Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu1 5 10 15Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Thr Tyr

20 25 30Thr Ile Ser Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45Gly Ile Ile Tyr Pro Ser Asp Ser Asp Thr Ile Tyr Ser Pro Ser Phe 50 55 60Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr65 70 75 80Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95Ala Arg Ser Ser Val Val Gly Phe Asp Val Trp Gly Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130 135 140Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser145 150 155 160Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 195 200 205Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Glu Phe Asp Tyr Lys 210 215 220Asp Asp Asp Asp Lys Gly Ala Pro His His His His His His225 230 235288214PRTArtificial Sequencesynthetic affinity ligand peptide sequence 288Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala Ala Ser Arg Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Leu Ser Trp Pro Glu 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu Ala 210

Claims

1-37. (canceled)

38. A method of selecting an affinity ligand that specifically binds to a target molecule under neutral buffer conditions and has reduced binding strength to the target molecular under mild elution conditions, the method comprising the steps of: a) expressing a naive affinity ligand library to produce a plurality of affinity ligands; b) providing a solid support linked to a target; c) contacting the solid support with the plurality of affinity ligands; d) washing the solid support with a wash buffer to remove unbound affinity ligands, wherein the wash buffer comprises neutral buffer conditions; e) contacting the solid support with an elution buffer comprising (i) a pH of about 4.0 to about 5.5 or (ii) about 1-2 M MgCl.sub.2; and f) identifying affinity ligands that substantially dissociate from the solid support in the elution buffer.

39. The method of claim 38, wherein the plurality of affinity ligands is encoded by a plurality of nucleic acid sequences.

40. The method of claim 39, wherein the plurality of nucleic acid sequences comprise a heterologous promoter operably linked thereto.

41. The method of claim 38, wherein the plurality of affinity ligands are expressed on a plurality of phage.

42. The method of claim 38, wherein the elution buffer comprises a pH of about 4.0 to about 5.5 and a relatively low salt concentration.

43. The method of claim 38, wherein the elution buffer comprises about 1 M to 2 M MgCl.sub.2 and a relatively neutral pH.

44. The method of claim 38, wherein the elution buffer comprises about 1 M to 2 M MgCl.sub.2 and a pH of about 6.0 to 8.0.

45. The method of claim 38, wherein the wash buffer comprises a pH of 6.0-8.0.

46. The method of claim 38, wherein the wash buffer comprises a relatively low salt concentration.

47. The method of claim 38, wherein the target is an immunoglobulin.

48. The method of claim 38, wherein the target is an immunoglobulin M (IgM), an immunoglobulin A (IgA), or an immunoglobulin E (IgE).

49. The method of claim 38, wherein the plurality of affinity ligands is a plurality of antibodies or derivatives thereof.

50. The method of claim 38, wherein the plurality of affinity ligands is a plurality of Fab fragments or derivatives thereof.

51. The method of claim 38, wherein the affinity ligand library is not preselected for characteristics favoring reduced binding strength to the target molecule under mild elution conditions.

52-58. (canceled)