Anti-Gliadin Antibodies

Abstract

The present invention provides anti-gliadin antibodies and antibody fragments, and polypeptides encoding the antibodies or fragment. Also disclosed are methods and Kits for the use of such antibodies, fragments, or polypeptides in detection of gliadin. Further provided are heavy chain and light chain variable sequences and associated sequences of complementarity-determining regions (CDRs).

Claims

1-18. (canceled)

19. An isolated nucleic acid comprising a vector, the vector comprising a polynucleotide sequence encoding a protein, the protein comprising an antibody heavy chain variable region comprising a CDR1 of SEQ ID NO:3, a CDR2 of SEQ ID NO:4, and a CDR3 of SEQ ID NO: 5; an antibody light chain variable region comprising a CDR1 of SEQ ID NO:8, a CDR2 of SEQ ID NO:9, and a CDR3 of SEQ ID NO:10; an antibody a heavy chain variable region comprising a CDR1 of SEQ ID NO: 13, a CDR2 of SEQ ID NO: 14, and a CDR3 of SEQ ID NO: 15; or an antibody light chain variable region comprising a CDR1 of SEQ NO:18, a CDR2 of SEQ ID NO:19, and a CDR3 of SEQ ID NO:20.

20. The nucleic acid of claim 19, wherein the polynucleotide sequence encodes a protein comprising (1) an antibody heavy chain variable region having the amino acid sequence of SEQ ID NO:2 or 12; or (2) an antibody light chain variable region having the amino acid sequence of SEQ ID NO:7 or 17.

21. The nucleic acid of claim 19, wherein the polynucleotide sequence is SEQ ID NO:1, 6, 11, or 16.

22. The nucleic acid of claim 19, further comprising a promoter operably linked to the polynucleotide sequence.

23. A cell comprising the nucleic acid of claim 19.

24. The cell of claim 23, comprising a bacterial cell.

25. The cell of claim 24, wherein the cell is an Escherichia coli cell.

26. The cell of claim 23, comprising a eukaryotic cell.

27. The cell of claim 26, wherein the cell is selected from a yeast cell, a COS cell, a CHO cell, a HeLa cell, and a myeloma cell.

28. A cell comprising the nucleic acid of claim 22.

29. The cell of claim 28, wherein the cell comprises the protein encoded by the nucleic acid.

30. A composition comprising an isolated antibody or antibody fragment comprising (1) a heavy chain variable region comprising a CDR1 of SEQ ID NO:3, a CDR2 of SEQ ID NO:4, and a CDR3 of SEQ ID NO:5 and a light chain variable region comprising a CDR1 of SEQ ID NO:8, a CDR2 of SEQ ID NO:9, and a CDR3 of SEQ ID NO:10; or (2) a heavy chain variable region comprising a CDR1 of SEQ ID NO:13, a CDR2 of SEQ ID NO:14, and a CDR3 of SEQ ID NO: 15 and a light chain variable region comprising a CDR1 of SEQ ID NO: 18, a CDR2 of SEQ ID NO:19, and a CDR3 of SEQ ID NO:20, wherein the antibody or antibody fragment binds to gliadin at an epitope defined by the amino acid sequence set forth in any one of SEQ ID NOs:21-32, wherein the antibody or antibody fragment is linked to a solid support.

31. The composition of claim 30, comprising an antibody or antibody fragment wherein the heavy chain variable region has an amino acid sequence of SEQ ID NO:2, and wherein the light chain variable region has an amino acid sequence of SEQ ID NO:7.

32. The composition of claim 30, comprising an antibody or antibody fragment wherein the heavy chain variable region has an amino acid sequence of SEQ ID NO: 12, and wherein the light chain variable region has an amino acid sequence of SEQ ID NO: 17.

33. The composition of claim 30, wherein the solid support is one of a microtiter plate, a microchip, or a bead.

34. A composition comprising a first isolated antibody or antibody fragment, and a second isolated antibody or antibody fragment, wherein the first and second isolated antibodies or antibody fragments comprise, respectively (1) a heavy chain variable region comprising a CDR1 of SEQ ID NO:3, a CDR2 of SEQ ID NO:4, and a CDR3 of SEQ ID NO:5 and a light chain variable region comprising a CDR1 of SEQ ID NO:8, a CDR2 of SEQ ID NO:9, and a CDR3 of SEQ ID NO:10; and (2) a heavy chain variable region comprising a CDR1 of SEQ ID NO:13, a CDR2 of SEQ ID NO:14, and a CDR3 of SEQ ID NO: 15 and a light chain variable region comprising a CDR1 of SEQ ID NO: 18, a CDR2 of SEQ ID NO:19, and a CDR3 of SEQ ID NO:20, wherein the first and second antibodies or antibody fragments bind to gliadin at an epitope defined by the amino acid sequence set forth in any one of SEQ ID NOs:21-32, wherein the first and second antibodies or antibody fragments are linked to a solid support.

35. The composition of claim 34, wherein the first and second isolated antibodies or antibody fragments comprise, respectively (1) the heavy chain variable region having an amino acid sequence of SEQ ID NO:2, and the light chain variable region having an amino acid sequence of SEQ ID NO:7; and (2) the heavy chain variable region having an amino acid sequence of SEQ ID NO: 12, and the light chain variable region having an amino acid sequence of SEQ ID NO: 17.

36. The composition of claim 34, wherein the solid support is one of a microtiter plate, a microchip, or a bead.

37. The composition of claim 35, wherein the solid support is one of a microtiter plate, a microchip, or a bead.

37. A kit comprising the composition of claim 30.

38. A kit comprising the composition of claim 34.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0071] FIG. 1: The DNA and amino acid sequences of the heavy and light chain variable regions of antibody 13F6 (SEQ ID NOS:34-37, respectively).

[0072] FIG. 2: The DNA and amino acid sequences of the heavy and light chain variable regions of antibody 14G11 (SEQ ID NOS:38-41, respectively).

[0073] FIG. 3: Indirect ELISA results from 13F6 and 14G11 clones against gluten and non-gluten proteins. All antigens were coated overnight at 1 μg/ml, 100 μl in each well. After standard blocking and washing procedures, antibodies were added at 10 ng/ml, in 100 and plates incubated at 25° C. for 1 hour. After washing steps, anti-mouse HRP were added and incubated for 1 hour, followed by TMB addition for 6 minutes. After acid quenching, absorbance was measured at 450 nm.

[0074] FIG. 4: Indirect ELISA results from antibodies 13F6 (a) and 14G11 (b) as well as the commercial R5 (c) antibody against wheat, barley, rye, oat, and 33-mer. All antigens were coated overnight at 20 μg/ml, 100 μl in each well. After standard blocking and washing procedures, antibodies was added at 0.8-20 ng/ml, in 100 μl, and incubated at 25° C. for 1 hour. After washing steps, anti-mouse HRP was added and plates were incubated for 1 hour, followed by TMB (peroxidase substrate) addition and further incubation for 5 minutes. After acid quenching, absorbance was measured at 450 nm.

[0075] FIG. 5: SDS-Page and Western Blot data for R5, 14G11, 13F6, IgG2b and IgG1 control against 33-mer conjugated BSA, and PWG gliadins.

[0076] FIG. 6: Results of affinity measurement for 14G11 and 13F6 using Biacore T200 software. Fitting Model: 1:1 binding.

[0077] FIG. 7: Binding characteristics of 13F6 and 14G11 in comparison with R5. Antigens coating: Barley, Rye, Wheat, 33-mer, Oat, 1 ug/mL in 50 mM Carbonate buffer pH 9.4, 4° C., overnight. Individual gliadins: Glaidin (PWG), 5 mg/mL, 60% EtOH, Barley and Rye (Aromalab.de), 5 mg/mL, 60% EtOH Oat mixture, 4 ug/mL 60% EtOH. 1 mg/mL of each of four oats: Oat1 Clav950, Oat0 AV06ID, Oat2, 168069, Oat5, 168069, Blocking: SEBB, 300 uL/well, RT, 1.5 hour. 1st Ab (43G2, R5, 13F6 and 14G11): 20-08 ng/mL in SEDB, 100 uL/well, RT, 1 hour. 2nd Ab: goat anti-mouse IgG Fc (Jackson Lab Cat#115-035-008) 160 ng/mL in SEDB, 100 uL/well, RT, 1 hour TMB: RT, 5 minutes. Notes: Desired response: wheat, barley, rye, 33mer. Undesired response: oat and others.

DETAILED DESCRIPTION

I. Anti-Gliadin Antibodies

[0078] Due to the significant rise in the instances of gluten-intolerance during the recent years, there is a corresponding increased need for developing a rapid and reliable means of detecting the presence of gluten in food or beverage so as to permit individuals having gluten-sensitivity to determine, in a real-world sense, their food or drink choices. The present invention provides novel anti-gliadin antibodies that can be used in a kit and method for rapid detection of gluten with high sensitivity and accuracy.

[0079] In some embodiments, the present invention provides anti-gliadin antibodies that recognize a previously undefined epitope of gliadin. This newly defined epitope is an amino acid sequence comprising SEQ ID NO:21, for example, represented by the amino acid sequence set forth in any one of SEQ ID NOs:22-29. In some examples, the antibody includes CDRs 1, 2, and 3 of the V.sub.H region (i.e., SEQ ID NOs:3, 4, 5, and 2, respectively) and CDRs 1, 2, and 3 of the V.sub.L region (i.e., SEQ ID NOs:8, 9, 10, and 7, respectively) of the 13F6 antibody as those CDRs are shown in FIG. 1. In some embodiments, the invention provides anti-gliadin antibodies having CDRs 1, 2, and 3 of the V.sub.H region (i.e., SEQ ID NOs:13, 14, 15, and 12, respectively) and CDRs 1, 2 and 3 of the V.sub.L region (i.e., SEQ ID NOs:18, 19, 20, and 17, respectively) of the 14G11 antibody as those CDRs are shown in FIG. 2.

[0080] While the exemplary antibodies, 13F6 and 14G11, have the amino acid sequence of heavy chain variable domains set forth in SEQ ID NO:2 and SEQ ID NO:12, respective, and the amino acid sequence of light chain variable domains set forth in SEQ ID NO:7 and SEQ ID NO:17, respectively, it is well known to those of skill in the art that the same antibody binding characteristics can be conserved by maintaining the same CDRs in the heavy and light chains while the framework regions is modified (e.g., humanized). Furthermore, single chain antibodies having essentially the same antigen-binding characteristics can be made by fusion of the matching V.sub.H and V.sub.L of an antibody (e.g., 13F6 or 14G11) without including some or all of the constant regions of the heavy and light chains. The exemplary DNA sequences encoding the heavy and light chains of the anti-gliadin antibodies are provided in FIGS. 1 and 2, however, alternative DNA coding sequences may be used to recombinantly produce the same antibodies due to codon-wobbling.

[0081] The anti-gliadin antibodies of the present invention not only recognize previously undefined gliadin epitope, an amino acid sequence no longer than 8-10 amino acids comprising SEQ ID NO:21 (e.g., the epitope having the amino acid sequence of any one of SEQ ID NO:22-31), they also exhibit surprisingly high level of antigen affinity compared to known gliadin antibodies such as R5, G12, and A1, etc. See, e.g., Osman et al., J. Gastroenteroloy & Hepatology 2001, 13:1189-1193; Moron et al., PLoS ONE 2008, 3(5): e2295, 1-13. Typically the anti-gliadin antibodies of the present invention exhibit a KD in binding with wheat gliadin less than about 200 nM, about 100 nM, or about 50 nM, for example, between about 1-100 nM, about 2-50 nM, about 4-50 nM, or about 5-50 nM.

II. Production of Immunoconjugates

[0082] The anti-gliadin antibodies of the invention can be linked to detectable molecules (DM) through the DM carboxyl terminus, the DM amino terminus, through an interior amino acid residue of the DM such as cysteine, or any combination thereof. Similarly, the DM can be linked directly to heavy, light, Fc (constant region) or framework regions of the antibody. Linkage can occur through the antibody's amino or carboxyl termini, or through an interior amino acid residue. Further, multiple DM molecules (e.g., any one of from 2-10) can be linked to the anti-gliadin antibody and/or multiple antibodies (e.g., any one of from 2-5) can be linked to an DM. The antibodies used in a multivalent immunoconjugate composition of the present invention can be directed to the same or different gliadin epitopes. In addition to a covalent linkage, a non-covalent linkage (e.g., via physical adsorption) can be used in making the immunoconjugate as well.

[0083] Immunoconjugates include, but are not limited to, molecules in which there is a detectable agent linked to an antibody via a covalent linkage or non-covalent linkage (e.g., by way of physical adsorption). A detectable agent is an agent having the capability of generating a detectable signal, e.g., radioactive, colormetric, fluorescent, time-resolved fluorescence, luminescence, electrical, electrochemical, or electromagnetic signal.

[0084] A. Recombinant Methods

[0085] The nucleic acid sequence encoding a immunoconjugate comprising the anti-gliadin antibody of the present invention and a detectable moiety can be prepared by any suitable method including, for example, cloning of appropriate sequences or by direct chemical synthesis by methods such as the phosphotriester method of Narang, et al., Meth. Enzymol. 68:90-99 (1979); the phosphodiester method of Brown, et al., Meth. Enzymol. 68:109-151 (1979); the diethylphosphoramidite method of Beaucage, et al., Tetra. Lett. 22:1859-1862 (1981); the solid phase phosphoramidite triester method described by Beaucage & Caruthers, Tetra. Letts. 22(20):1859-1862 (1981), e.g., using an automated synthesizer as described in, for example, Needham-VanDevanter, et al. Nucl. Acids Res. 12:6159-6168 (1984); and, the solid support method of U.S. Pat. No. 4,458,066. Chemical synthesis produces a single stranded oligonucleotide. This may be converted into double stranded DNA by hybridization with a complementary sequence, or by polymerization with a DNA polymerase using the single strand as a template. One of skill would recognize that while chemical synthesis of DNA is limited to sequences of about 100 bases, longer sequences may be obtained by the ligation of shorter sequences.

[0086] In a preferred embodiment, the nucleic acid sequences of this invention are prepared by cloning techniques. Examples of appropriate cloning and sequencing techniques, and instructions sufficient to direct persons of skill through many cloning exercises are found in Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL (3RD ED.), Vols. 1-3, Cold Spring Harbor Laboratory (2001)), Berger and Kimmel (eds.), GUIDE TO MOLECULAR CLONING TECHNIQUES, Academic Press, Inc., San Diego Calif. (1987)), or Ausubel, et al. (eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing and Wiley-Interscience, NY (1987-2009). Product information from manufacturers of biological reagents and experimental equipment also provide useful information. Such manufacturers include the SIGMA chemical company (Saint Louis, Mo.), R&D systems (Minneapolis, Minn.), Pharmacia LKB Biotechnology (Piscataway, N.J.), CLONTECH Laboratories, Inc. (Palo Alto, Calif.), Chem Genes Corp., Aldrich Chemical Company (Milwaukee, Wis.), Glen Research, Inc., GIBCO BRL Life Technologies, Inc. (Gaithersberg, Md.), Fluka Chemica-Biochemika Analytika (Fluka Chemie AG, Buchs, Switzerland), Invitrogen, San Diego, Calif., and Applied Biosystems (Foster City, Calif.), as well as many other commercial sources known to one of skill.

[0087] Nucleic acids encoding an anti-gliadin antibody or binding fragment thereof or the detectable molecule (DM) can be modified to form the immunoconjugates of the present invention. Modification by site-directed mutagenesis is well known in the art. Nucleic acids encoding DM or anti-gliadin antibodies can be amplified by in vitro methods. Amplification methods include the polymerase chain reaction (PCR), the ligase chain reaction (LCR), the transcription-based amplification system (TAS), the self-sustained sequence replication system (3 SR). A wide variety of cloning methods, host cells, and in vitro amplification methodologies are well known to persons of skill.

[0088] In one embodiment, immunoconjugates are prepared by inserting the cDNA which encodes an anti-gliadin scFv antibody into a vector which comprises the cDNA encoding the DM. The insertion is made so that the scFv and the DM are read in frame, that is in one continuous polypeptide containing a functional Fv region and a functional DM region.

[0089] Once the nucleic acids encoding a DM, anti-gliadin antibody, or an immunoconjugate of the present invention are isolated and cloned, one may express the desired protein in a recombinantly engineered cell such as bacteria, plant, yeast, insect and mammalian cells. It is expected that those of skill in the art are knowledgeable in the numerous expression systems available for expression of proteins including E. coli, other bacterial hosts, yeast, and various higher eucaryotic cells such as the COS, CHO, HeLa and myeloma cell lines. No attempt to describe in detail the various methods known for the expression of proteins in prokaryotes or eukaryotes will be made. In brief, the expression of natural or synthetic nucleic acids encoding the isolated proteins of the invention will typically be achieved by operably linking the DNA or cDNA to a promoter (which is either constitutive or inducible), followed by incorporation into an expression cassette. The cassettes can be suitable for replication and integration in either prokaryotes or eukaryotes. Typical expression cassettes contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the DNA encoding the protein. To obtain high level expression of a cloned gene, it is desirable to construct expression cassettes which contain, at the minimum, a strong promoter to direct transcription, a ribosome binding site for translational initiation, and a transcription/translation terminator. For E. coli this includes a promoter such as the T7, trp, lac, or lambda promoters, a ribosome binding site and preferably a transcription termination signal. For eukaryotic cells, the control sequences can include a promoter and preferably an enhancer derived from immunoglobulin genes, SV40, cytomegalovirus, and a polyadenylation sequence, and may include splice donor and acceptor sequences. The cassettes of the invention can be transferred into the chosen host cell by well-known methods such as calcium chloride transformation or electroporation for E. coli and calcium phosphate treatment, electroporation or lipofection for mammalian cells. Cells transformed by the cassettes can be selected by resistance to antibiotics conferred by genes contained in the cassettes, such as the amp, gpt, neo and hyg genes.

[0090] One of skill would recognize that modifications can be made to a nucleic acid encoding a polypeptide of the present invention (i.e., anti-gliadin antibody an immunoconjugate formed from the combination of the antibody and a DM) without diminishing its biological activity. Some modifications may be made to facilitate the cloning, expression, or incorporation of the targeting molecule into a fusion protein. Such modifications are well known to those of skill in the art and include, for example, termination codons, a methionine added at the amino terminus to provide an initiation, site, additional amino acids placed on either terminus to create conveniently located restriction sites, or additional amino acids (such as poly His) to aid in purification steps.

[0091] In addition to recombinant methods, the immunoconjugates, DM, and antibodies of the present invention can also be constructed in whole or in part using standard peptide synthesis. Solid phase synthesis of the polypeptides of the present invention of less than about 50 amino acids in length may be accomplished by attaching the C-terminal amino acid of the sequence to an insoluble support followed by sequential addition of the remaining amino acids in the sequence. Techniques for solid phase synthesis are described by Barany & Merrifield, THE PEPTIDES: ANALYSIS, SYNTHESIS, BIOLOGY. VOL. 2: SPECIAL METHODS IN PEPTIDE SYNTHESIS, PART A. pp. 3-284; Merrifield, et al. J. Am. Chem. Soc. 85:2149-2156 (1963), and Stewart, et al., SOLID PHASE PEPTIDE SYNTHESIS, 2ND ED., Pierce Chem. Co., Rockford, Ill. (1984). Proteins of greater length may be synthesized by condensation of the amino and carboxyl termini of shorter fragments. Methods of forming peptide bonds by activation of a carboxyl terminal end (e.g., by the use of the coupling reagent N, N′-dicycylohexylcarbodiimide) are known to those of skill.

[0092] B. Purification

[0093] Once expressed, the recombinant immunoconjugates, antibodies, and/or detectable molecules of the present invention can be purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, and the like (see, generally, R. Scopes, PROTEIN PURIFICATION, Springer-Verlag, N.Y. (1982)). Substantially pure compositions of at least about 90 to 95% homogeneity are preferred, and 98 to 99% or more homogeneity are most preferred for diagnostic uses.

[0094] Methods for expression of single chain antibodies and/or refolding to an appropriate active form, including single chain antibodies, from bacteria such as E. coli have been described and are well-known and are applicable to the antibodies of this invention. See, Buchner, et al., Anal. Biochem. 205:263-270 (1992); Pluckthun, Biotechnology 9:545 (1991); Huse, et al., Science 246:1275 (1989) and Ward, et al., Nature 341:544 (1989), all incorporated by reference herein.

[0095] Often, functional heterologous proteins from E. coli or other bacteria are isolated from inclusion bodies and require solubilization using strong denaturants, and subsequent refolding. During the solubilization step, as is well-known in the art, a reducing agent must be present to separate disulfide bonds. An exemplary buffer with a reducing agent is: 0.1 M Tris pH 8, 6 M guanidine, 2 mM EDTA, 0.3 M DTE (dithioerythritol). Reoxidation of the disulfide bonds can occur in the presence of low molecular weight thiol reagents in reduced and oxidized form, as described in Saxena, et al., Biochemistry 9: 5015-5021 (1970), incorporated by reference herein, and especially as described by Buchner, et al., supra.

[0096] Renaturation is typically accomplished by dilution (e.g., 100-fold) of the denatured and reduced protein into refolding buffer. An exemplary buffer is 0.1 M Tris, pH 8.0, 0.5 M L-arginine, 8 mM oxidized glutathione (GSSG), and 2 mM EDTA.

[0097] As a modification to the two chain antibody purification protocol, the heavy and light chain regions are separately solubilized and reduced and then combined in the refolding solution. A preferred yield is obtained when these two proteins are mixed in a molar ratio such that a 5 fold molar excess of one protein over the other is not exceeded. It is desirable to add excess oxidized glutathione or other oxidizing low molecular weight compounds to the refolding solution after the redox-shuffling is completed.

[0098] C. Detectable Labels

[0099] In some embodiments, the antibodies of the invention or a binding fragment thereof or a polypeptide comprising the antibody or fragment can be coupled to detectable labels. The linkage can be covalent or non-covalent. Detectable labels suitable for such use include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Useful labels in the present invention include magnetic beads (e.g. DYNABEADS), fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, green fluorescent protein, and the like), radiolabels (e.g., .sup.3H, .sup.125I, .sup.35S, .sup.14C, or .sup.32P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and colorimetric labels such as colloidal gold or colored glass or plastic (e.g. polystyrene, polypropylene, latex) beads.

[0100] Means of detecting such labels are well known to those of skill in the art. Thus, for example, radiolabels may be detected using photographic film or scintillation counters, fluorescent markers may be detected using a photodetector to detect emitted illumination. Enzymatic labels are typically detected by providing the enzyme with a substrate and detecting the reaction product produced by the action of the enzyme on the substrate, and colorimetric labels are detected by simply visualizing the colored label.

[0101] D. Conjugation to the Antibody

[0102] In a non-recombinant embodiment of the invention, detectable molecules are linked to the anti-gliadin antibodies of the present invention using any number of means known to those of skill in the art. Both covalent and noncovalent attachment means may be used with anti-gliadin antibodies of the present invention.

[0103] The procedure for attaching an effector molecule to an antibody will vary according to the chemical structure of the DM. Polypeptides typically contain a variety of functional groups; e.g., carboxylic acid (COOH), free amine (—NH.sub.2) or sulfhydryl (—SH) groups, which are available for reaction with a suitable functional group on an antibody to result in the binding of the effector molecule.

[0104] Alternatively, the antibody is derivatized to expose or to attach additional reactive functional groups. The derivatization may involve attachment of any of a number of linker molecules, such as those available from Pierce Chemical Company (Rockford Ill.).

[0105] A “linker”, as used herein, is a molecule that is used to join the antibody to the effector molecule. The linker is capable of forming covalent bonds to both the antibody and to the effector molecule. Suitable linkers are well known to those of skill in the art and include, but are not limited to, straight or branched-chain carbon linkers, heterocyclic carbon linkers, or peptide linkers. Where the antibody and the detectable molecule are polypeptides, the linkers may be joined to the constituent amino acids through their side groups (e.g., through a disulfide linkage to cysteine). In some embodiments, the linkers may be joined to the alpha carbon amino and carboxyl groups of the terminal amino acids.

[0106] In view of the large number of methods that have been reported for attaching a variety of fluorescent/chemiluminescent compounds, enzymes, dyes, and other agents (e.g., latex particles, nanoparticles, nanocrystals, colloidal gold, etc.) to antibodies one skilled in the art will be able to determine a suitable method for attaching a given agent to an antibody or other polypeptide.

III. Kits and Uses

[0107] In another embodiment, this invention provides for kits for the detection of gliadin or an immunoreactive fragment thereof, (i.e., collectively, a “gliadin protein”) in a food or beverage sample. A “food or beverage sample” as used herein is a sample of any substance intended for human consumption in the form of solid, semi-solid, or liquid that potentially could contain gliadin.

[0108] Kits will typically comprise an anti-gliadin antibody or antibody fragment of the present invention, the embodiments being as described herein. In some embodiments, the anti-gliadin antibody or antibody fragment will be an anti-gliadin Fv fragment, such as a scFv fragment, or a recombinant polypeptide comprising an anti-gliadin antibody or a gliadin-binding fragment thereof plus a heterologous amino acid sequence.

[0109] In addition the kits will typically include instructional materials disclosing means of use of an antibody of the present invention (e.g. for detection of gliadin in a sample). The kits may also include additional components to facilitate the particular application for which the kit is designed. Thus, for example, the kit may additionally contain means of detecting the label (e.g. enzyme substrates for enzymatic labels, filter sets to detect fluorescent labels, appropriate secondary labels such as a sheep anti-mouse-HRP, or the like). The kits may additionally include buffers and other reagents routinely used for the practice of a particular method. In some cases, a positive control (a gliadin-containing sample) may be included in the kit to ensure that that detection assay is operating correctly. Such kits and appropriate contents are well known to those of skill in the art.

[0110] In one embodiment of the present invention, the gliadin-detection kit comprises an immunoassay. As described above, although the details of the immunoassays of the present invention may vary with the particular format employed, the method of detecting gliadin in a food or beverage sample generally comprises the steps of contacting the sample with an antibody that specifically reacts, under immunologically reactive conditions, to gliadin. The antibody is allowed to bind to gliadin under immunologically reactive conditions, and the presence of the bound antibody is detected directly or indirectly. The anti-gliadin antibody may be used, for example, as the capture antibody of an ELISA, or as a second antibody to bind to gliadin captured by the capture antibody. In some embodiments, the kits comprise an antibody or antibody fragment pre-bound to a solid support, e.g., a microchip, a microtiter plate or a bead. As is known in the art, the presence of the second antibody is typically then detected.

[0111] The antibodies provided herein are useful as diagnostic agents and in in vitro assays to detect the presence of gliadin in test samples. For example, the antibodies 13F6 and 14G11 and variants of these antibodies as described herein can be used as the targeting moieties of immunoconjugates in immunohistochemical assays to determine whether a sample contains gliadin. If the sample is one taken from a food item that is in solid form, processing steps of grinding and mixing/solubilizing may be necessary before the immunoassay can be conducted. As such, the kit optionally contains a solution suitable for dissolving the ground food sample before an immunoassay can be performed, see, e.g., U.S. patent application Ser. No. 15/065,198, U.S. Patent Application Publication No. US20140295406A1.

EXAMPLES

[0112] The following examples are provided by way of illustration only and not by way of limitation. Those of skill in the art will readily recognize a variety of non-critical parameters that could be changed or modified to yield essentially the same or similar results.

Example 1: Production and Functional Analysis of 13F6 and 14G11

[0113] Two clones of anti-gliadin antibodies, 13F6 and 14G11, have been identified as exhibiting high and comparable responses to barley, wheat, rye and a 33-mer amino acid sequence derived from the wheat gliadin protein (SEQ ID NO:33) (see, e.g., Shan et al., Science 297:2275-2279, 2002; Moron et al., PLoS ONE, 3(5):e2994:1-13, 2008; Moron et al., Am. J. Clin. Nutr. 87:405-414, 2008). 13F6 is an IgG2b antibody, whereas 14G11 is an IgG1 antibody.

[0114] Monoclonal antibodies 14G11 and 13F6 were raised by immunizing five (5) 8-9 week old female BALB/c mice with a 33-mer gluten specific peptide (amino acid sequence LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF (SEQ ID NO:33), Shan et al., Science 297:2275-2279, 2002) conjugated to KHL (Keyhole limpet hemocyanin), as well as gluten prolamins using standard immunization procedure. Serum samples obtained from blood draws were used to monitor titers of target specific antibodies in animals by indirect ELISA using wheat, barley, rye prolamins and unconjugated gluten specific peptide. Two animals with the highest serum titers were given the final immunization with 10 μg of gluten specific peptide in PBS intravenously three days before spleen harvest. Spleens of these mice were removed, spleen cells were extracted and fused with P3X63Ag8.653 mouse myeloma cells (ATCC, CRL-1580). Hybridoma cells were generated following standard protocols. Then hybridoma supernatants were collected and screened by indirect ELISA for the reactivity against gluten specific immunogenic peptide, barley, wheat, rye, oat, rice, corn and soy proteins. Purified antibodies were produced in protein A chromatography.

[0115] Specifically, both 13F6 and 14G11 show comparable responses to wheat, barley and rye proteins, but do not respond to other non-gluten grains, especially oat (FIGS. 3 and 4). This is highly desirable because oat, while similar to gluten, is not actually a gluten protein. Some other commercial assays (Romer Labs, or GlutenTox) use antibodies such as G12 and A1(Morón, B., Á. Cebolla, H. Manyani, M. Álvarez-Maqueda, M. Megias, M. del Carmen Thomas, M. C. López, and C. Sousa, Sensitive detection of cereal fractions that are toxic to celiac disease patients by using monoclonal antibodies to a main immunogenic wheat peptide. The American journal of clinical nutrition, 2008. 87(2): p. 405-414), which cross-react with oat and are therefore not ideal for gluten detection. In addition, 13F6 and 14G11 are much more sensitive to all gluten types than the well-accepted gold standard, the R5 antibody (Hernando, A., J. R. Mujico, D. Juanas, and E. Méndez, Confirmation of the Cereal Type in Oat Products Highly Contaminated with Gluten. Journal of the American Dietetic Association, 2006. 106(5): p. 665; Kahlenberg, F., D. Sanchez, I. Lachmann, L. Tuckova, H. Tlaskalova, E. Méndez, and T. Mothes, Monoclonal antibody R5 for detection of putatively coeliac-toxic gliadin peptides. European Food Research and Technology, 2006. 222(1-2): p. 78-82) (FIG. 4C). R5 is obtained from the Spanish National Center for Biology via Operon (website: operon.es). See FIG. 7.

Example 2: Affinity Studies

1. Objective

[0116] The aim of the current study is to measure the binding affinities between glutenin and three monoclonal antibodies using Biacore T200.

2. Materials

[0117] Test articles are listed below in Table 1 below:

TABLE-US-00003 TABLE 1 Samples MW (kDa) Concentration (mg/ml) Gluten Obtained from PWG group. 10 It is a gluten mixture.** Based on MS, 31.7 kDa was used to calculate molar concentration 14G11 ~140 kDA estimated from SDS-PAGE 4.9 13F6 5.2 R5 8 **PWG-gliadin is a reference material that has been produced under the guidance of the Prolamin Working Group (PWG). Its isolation and characterization is described in detail in van Eckert et al. (2006). Briefly, PWG-gliadin has been extracted from a mixture of 28 European wheat cultivars. Albumins and globulins were eliminated by extraction using 0.4M NaCl solution and gliadins were extracted with 60% ethanol. The gliadin extracts were concentrated, desalted by ultrafiltration, freeze-dried, and homogenized. The residual material after lyophilization is referred to as PWG-gliadin.

Biacore T200 (GE Healthcare)

[0118] Series S Sensor Chip CM5 (GE Healthcare Cat. No. BR-1005-30 Lot No.:10229292)
Capture antibody: Anti-mouse Fc gamma specific antibody, Cat. No. 115-005-071 (Jackson ImmunoResearch), 20 μg/ml in 10 mM Na-acetate pH 5.0

NHS: 100 mM N-hydroxysuccinimide in H.SUB.2.O

[0119] EDC: 400 mM 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide in H.sub.2O
Ethanolamine: 1 M ethanolamine hydrochloride, adjusted to pH 8.5 with NaOH
Running buffer HBS-EP: 10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% Tween-20

50 mM HCl

10 mM Glycine-HCl, pH 2.0

50 mM HCl, 3 M MgCl.SUB.2

3. Experimental Procedures

3.1 Glutenin Immobilized Through Amine Coupling

[0120] In this experiment, glutenin protein was coated through amine coupling method, the antibodies were flowed over the sensor chip as analytes. The experiment was carried out using the following protocol.

[0121] 3.1.1 Covalent Coupling of Glutenin

[0122] Coat glutenin onto the Series S Sensor Chip CM5 via primary amine groups using the following conditions: [0123] (1) Equilibrate: HBS-EP, flow rate 10 μl/min, 5 min; [0124] (2) Activate surface: inject NHS+EDC 1:1 mixture, flow rate 10 μl/min, 7 min; [0125] (3) Couple ligand: inject glutenin (15 μg/ml, 10 mM sodium acetate, pH 4.5), flow rate 10 μl/min, until ˜300 RU of glutenin is coated; [0126] (4) Deactivate excess reactive groups: inject ethanolamine, flow rate 10 μl/min, 7 min.

[0127] 3.1.2 Affinity Measurement

[0128] Test the pair-wise binding of test articles to the glutenin using the following assay setup: [0129] (1) Stabilize surface: Perform three start-up cycles with a dummy sample (HBS-EP buffer); [0130] (2) Equilibrate: flow running buffer over all flow cells for 1.5 hours; [0131] (3) Associate: inject the antigen at the lowest concentrations (see Parameter Summary Table below) over all flow cells, flow rate 30 μl/min, 14G11 and 13F6, 5 min and R5, 2.5 min; [0132] (4) Dissociate: flow running buffer over all flow cells, flow rate 30 μl/min, 14G11 and 13F6, 15 min and R5, 2.5 min; [0133] (5) Regenerate surface: flow regeneration buffer (14G11 and 13F6, 10 mM Glycine-HCl and R5, 50 mM HCl, 3 M MgCl.sub.2) 3 times, flow rate 100 μl/min, 15 sec; [0134] (6) Increase the analyte concentration by 2 or 3 fold, repeat steps (1) through (5). All together there were five analyte concentrations and one repeat of a medium concentration (see Parameter Summary Table 2 below). The curve of the repeat cycle should coincide with that of the previous cycle to confirm that the regeneration condition was appropriate.

TABLE-US-00004 TABLE 2 Immobilization Analyte 14G11 13F6 R5 Ligand Glutenin Immobilization 283.8 level (RU) Association and Dissociation Flow rate (μl/min) 30  Association time (s) 300 300 150 Dissociation time (s) 600 600 150 Sample Concentration Concentration (nM) 3, 9, 27(x2), 3, 9(x2), 27, 20, 40, 80(x2), 81, 243 81, 243 160, 320 Regeneration Flow rate (μl/min) 100   Regeneration 15(x3) contact time (s) Regeneration 10 mM 10 mM 50 mM HCl, buffer Glycine-HCl Glycine-HCl 3M MgCl.sub.2

4. Results and Discussion

[0135] The experimental data were processed, and fitted locally to 1:1 interaction model in Biacore T200 evaluation software. The model describes interaction A+B=AB, is the simplest model for kinetic evaluation, therefore is recommended as default unless there is good reason to choose a different model.

[0136] The experimental result is summarized in FIG. 6.

[0137] The curves of medium concentrations coincided, suggesting that the regeneration conditions were good. The gluten surface was not damaged after several cycles of regeneration. The Chi′ were about or below 10% of the Rmax, which means that the fitting of experimental data to 1:1 interaction model was not perfect but reliable. The U-values of all three antibodies were low, <5. All in all, the curve fitting is good enough and the result is reliable. The result should better reflect the real binding affinity between gluten and antibodies.

5. Summary

[0138] In this study, the binding affinities between glutenin and three monoclonal antibodies were determined. The experiment was carried out where glutenin was amine coupled to the sensor chip and antibodies were used as analytes. The result of is with good curve fitting and low U-values, and thus is reliable.

Example 3: Binding Validation and Epitope Mapping

Materials

Antigen: Modified Gliadin(PWG)

[0139] 6SEN 33-Mer peptide Unconjugated,33-mer peptide (5 mg/ml) [0140] 6SEN 33-Mer peptide BSA-conjugated, BSA-33 mer peptide(5 mg/ml)
Antibody: R5 monoclonal Ab (8 mg/ml) [0141] 14G11 monoclonal Ab (4.94 mg/ml) [0142] 13F6 monoclonal Ab (5.2 mg/ml)
Other reagents and solutions: [0143] PBS buffer: NaCl 137 mM; KCl 2.7 mM; Na.sub.2HPO.sub.4 4.3 mM; KH.sub.2PO.sub.4 1.4 mM, pH 7.4

[0144] PBS-T buffer: PBS buffer with 0.05% Tween 20 [0145] Coating buffer: 0.05 M NaHCO.sub.3, pH 9.6 [0146] Blocking Buffer: PBS buffer with 5% skimmed milk [0147] TMB [0148] 1 M HCl [0149] Goat anti-mouse IgG antibody(H+L)[HRP], GenScript [0150] IRDye800CW goat anti-mouse IgG (H+L), LI-COR

TABLE-US-00005 TABLE 3 ELISA condition Ag Coating BSA (negative ctrl)/BSA-Peptide/33-mer Peptide/Modified Gliadin (PWG) (targets) 10 μg/ml incubated at 4° C. overnight Blocking 5% MPBS, incubated at RT for 1.5 h Primary Ab diluted Ab 10x starting from 10 μg/ml in 0.05% PBST, incubated at 4° C. for 1.5 h Secondary Ab Goat anti-mouse IgG [HRP] 0.1 μg/ml, incubated at 4° C. for 45 min Western blot condition Blocking 5% MPBS, incubated at RT for 1 h Primary Ab diluted mAb 0.5 μg/ml in 0.05% PBST, incubated at RT for 1.5 h Secondary Ab IRDye 800CW goat anti mouse IgG [HRP] 0.1 μg/ml, incubated at RT for 45 min

[0151] All three mAbs bound the target specifically.

[0152] The binding affinity of R5 is lower than the other two mAbs, which is consistent with the result of affinity measurement by Biacore.

[0153] Antigen1 is a protein of 296 amino acids.

TABLE-US-00006 (SEQ ID NO: 42) MKTFLILALL AIVATTATTA VRVPVPQPQP QNPSQPQPQR QVPLVQQQQF PGQQQQFPPQ QPYPQPQPFP SQQPYLQLQP FPQPQPFPPQ LPYPQPPPFS PQQPYPQPQP QYPQPQQPIS QQQAQQQQQQ QQQQQQQQQQ QQILPQILQQ QLIPCRDVVL QQHNIAHARS QVLQQSTYQP LQQLCCQQLW QIPEQSRCQA IHNVVHAIIL HQQQQQQQPS SQVSLQQPQQ QYPSGQGFFQ PSQQNPQAQG SVQPQQLPQF EEIRNLALQT LPRMCNVYIP PYCSTTTAPF GIFGTN 

[0154] Antigen2 is a protein of 290 amino acids.

TABLE-US-00007 (SEQ ID NO: 43) MVRVPVPQLQ PQNPSQQQPQ EQVPLVQQQQ FPGQQQPFPP QQPYPQPQPF PSQQPYLQLQ PFPQPQLPYP QPQLPYPQPQ LPYPQPQPFR PQQPYPQSQP QYSQPQQPIS QQQQQQQQQQ QQKQQQQQQQ QILQQILQQQ LIPCRDVVLQ QHSIAYGSSQ VLQQSTYQLV QQLCCQQLWQ IPEQSRCQAI HNVVHAIILH QQQQQQQQQQ QQPLSQVSFQ QPQQQYPSGQ GSFQPSQQNP QAQGSVQPQQ LPQFEEIRNL ALETLPAMCN VYIPPYCTIA PVGIFGTNYR

[0155] Antigen3 is a peptide of 33 amino acids.

TABLE-US-00008 (SEQ ID NO: 33) LQLQPFPQPQ LPYPQPQLPY PQPQLPYPQP QPF

[0156] Peptide library design: peptide length/overlapping/offset=10aa/7aa/3aa; Cys was replaced by Ser in the peptide library; N-terminal biotinylated; crude product.

TABLE-US-00009 TABLE 4 Epitope mapping screening-condition Epitope ELISA condition Coating Streptavidin (Anchor protein); 33-Mer Peptide (positive 10 μg/ml incubated at Ctrl); Modified Gliadin(PWG) 4° C. overnight (positive Ctrl) 10 μg/ml incubated at 4° C. overnight Blocking 5% MPBS, incubated at RT for 1.5 h Capture crude peptide 50 μg/ml each, / incubated at RT for 1.5 h Primary R5 2 μg/ml; 14G11, 13F6, IgG2b Ab control mAb, IgGl control mAb 0.5 μg/ml incubated at RT for 1.5 h, incubated at RT for 1.5 h Secondary Goat anti-mouse IgG [HRP] Ab 0.1 μg/ml, incubated at RT for 45 min

[0157] All peptides designed from antigen 1/2/3 are available except 42, AQQQQQQQQQ (SEQ ID NO:44).

[0158] The binding between peptide libraries and mAbs were assayed by ELISA. And isotype and blank PBS control binding was included.

[0159] ELISA against peptide libraries was done twice. The result is the same. The binding between mAbs and peptide binders identified from the libraries was done once again to confirm the result.

[0160] 13F6 and 14G11 seemed to recognize the same epitope. The ELISA OD450 values of 13F6 were slightly higher than those of 14G11, suggesting the binding affinity of 13F6 was slightly higher, which is consistent with the result of affinity measurement.

[0161] None of the peptides seemed to bind R5. Even at R5 concentration as high as 10 μg/ml, none of antigen 3 peptides or ‘QQPFP’ (SEQ ID NO:45)-containing peptides bound R5 (data not shown). The peptides used for this study were crude peptides with low purity, coupled with the fact that the binding affinity of R5 was extremely low, with KD of ˜0.2 This might result in this outcome.

[0162] Peptide 42 (AQQQQQQQQQ (SEQ ID NO:44)) is the only one peptide that is not available. Seeing that peptide 43 (QQQQQQQQQQ (SEQ ID NO:45)) did not bind any of the three mAbs and the sequence of peptide 42 is not similar to those of identified peptide binders, it is highly possible that the peptide would not bind the mAbs.

[0163] Based on the screening result, 13 peptides could be recognized by the 13F6 and 14G11 antibodies.

[0164] These 13 peptides all have a common amino acid sequence of ‘Q(Q/L)PYPQ.’ (SEQ ID NO:21) The stretch of Q(Q/L)PYPQ (SEQ ID NO:21)sequence is believed to be the core of epitopes of 13F6 and 14G11 antibodies. This is in contrast to epitope sequences of previously known anti-gliadin antibodies (A1: QLPYPQP (SEQ ID NO:26); R5: QQPFP (SEQ ID NO:45); G12: QPQLPY (SEQ ID NO:47); QPQQPY (SEQ ID NO:48); QPQLPF (SEQ ID NO:49)).

[0165] The OD450 values of group B were lower than those of group A, suggesting the C-terminal residue(s), e.g., P or PQ, contributed to antigen binding. See Table 5 below.

TABLE-US-00010 TABLE 5 Common Group OD450nm sequence Peptides A ~2.0(13F6) Q(Q/L)PYPQ PQQPYPQP PQPQLPYP QLPYPQPQ PQLPYPQP PPQQPYPQ ~1.0(14G11) (P/S) QP QP LP QP PQ (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 24) NO: 50) NO: 51) NO: 52) NO: 53) NO: 54) SPQQPYPQ QPQLPYPQ PQLPYPQP PQQPYPQS PPQLPYPQ PQ PQ QL QP PP (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 55) NO: 56) NO: 57) NO: 58) NO: 59) B <(13F6) PQ(Q/L)PYPQ PFPPQQPY YPQPQLPY PFRPQQPY <0.5(14G11) (SEQ ID PQ PQ PQ NO: 29) (SEQ ID (SEQ ID (SEQ ID NO: 60) NO: 61) NO: 62)

[0166] All patents, patent applications, and other publications, including GenBank Accession Numbers, cited in this application are incorporated by reference in the entirety for all purposes.

INFORMAL SEQUENCE LISTING

[0167]

TABLE-US-00011 13F6 Antibody Heavy Chain Variable Region DNA Sequence FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 CAGATCCAGTTGGTGCAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAGCATCTCCT GCAAGGCTTCTGGTTATACCTTCACAGACTATTCAATGCACTGGGTGAGGCAGGCTCCAGGAAA GGGTTTAAAGTGGATGGGCTGGATAAACACTGAGACTGGTGAGCCAACATATGCAGATGACTTC AAGGGACGATTTGCCTTCTCTTTGGAAACCTCTGCCAGCACTGCCTATCTGCAGATCAACAACC TCAAAAATGAGGACACGGCTACACATTTCTGTGCTCCAAGTGTTGCCTGGTTTGCTTACTGGGG CCAAGGGACTCTGGTCACTGTCTCTACA SEQ ID NO: 2 13F6 Antibody Heavy Chain Variable Region Amino Acid Sequence FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 QIQLVQSGPELKKPGETVKISCKASGYTFTDYSMHWVRQAPGKGLKWMGWINTETGEPTYADDF KGRFAFSLETSASTAYLQINNLKNEDTATHFCAPSVAWFAYWGQGTLVTVST SEQ ID NO: 3 13F6 Antibody Heavy Chain CDR1 Amino Acid Sequence DYSMH SEQ ID NO: 4 13F6 Antibody Heavy Chain CDR2 Amino Acid Sequence WINTETGEPTYADDFKG SEQ ID NO: 5 13F6 Antibody Heavy Chain CDR3 Amino Acid Sequence SVAWFAY SEQ ID NO: 6 13F6 Antibody Light Chain Variable Region DNA Sequence FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 GATGTTTTGATGACCCAAACTCCACTCTCCCTGTCTGTCAGTCTTGGAGATCAGGCCTCCATCT CTTGTAGATCTAGTCAGAGCATTGTACAGAGTAATGGAAACACCCATTTAGAATGGTTCTTACA GAAACCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCA GACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTG AGGATCTGGGAGTTTATTACTGTTTTCAAGGTTCACATGTTCCATTCACGTTCGGCTCGGGGAC AAAGTTGGAAATAAAA SEQ ID NO: 7 13F6 Antibody Light Chain Variable Region Amino Acid Sequence FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 DVLMTQTPLSLSVSLGDQASISCRSSQSIVQSNGNTHLEWFLQKPGQSPKLLIYKVSNRFSGVP DRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPFTFGSGTKLEIK SEQ ID NO: 8 13F6 Antibody Light Chain CDR1 Amino Acid Sequence RSSQSIVQSNGNTHLE SEQ ID NO: 9 13F6 Antibody Light Chain CDR2 Amino Acid Sequence KVSNRFS SEQ ID NO: 10 13F6 Antibody Light Chain CDR3 Amino Acid Sequence FQGSHVPFT SEQ ID NO: 11 14G11 Antibody Heavy Chain Variable Region DNA Sequence FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 CAGATCCAGTTGGTGCAGTCTGGACCTGAGATGAAGAAGCCTGGAGAGACAGTCAAGATTTTTT GCAAGGCTTCTGGTTATACCCTCACAGACTATTCATGCACTGGGTGAAGCAGGCTCCAGGAAA GGGTTTAAAGTGGATGGGCTGGATAAACACTGAGACTGGTGAGCCAACATATGCAGATGACTTC AAGGGACGGTGTGCCTTTTCTTTGGAAACCTCTGTCAGCACTGCCTTTTTGCAGATCAACAACC TCAAAAATGAGGACATGGGAACATATTTCTGTGCCTCCTCTGGGGCCTGGTTTAGTTACTGGGG CCAAGGGACTCTGGTCACTGTCTCTGCA SEQ ID NO: 12 14G11 Antibody Heavy Chain Variable Region Amino Acid Sequence FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 QIQLVQSGPEMKKPGETVKIFCKASGYTLTDYSMHWVKQAPGKGLKWMGWINTETGEPTYADDF KGRCAFSLETSVSTAFLQINNLKNEDMGTYFCASSGAWFSYWGQGTLVTVSA SEQ ID NO: 13 14G11 Antibody Heavy Chain CDR1 Amino Acid Sequence DYSMH SEQ ID NO: 14 14G11 Antibody Heavy Chain CDR2 Amino Acid Sequence WINTETGEPTYADDFKG SEQ ID NO: 15 14G11 Antibody Heavy Chain CDR3 Amino Acid Sequence SGAWFSY SEQ ID NO: 16 14G11 Antibody Light Chain Variable Region DNA Sequence FR1-CDR1-FR2-CDR2-FR3 -CDR3 -FR4 GATGTTTTGCTGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCT CTTGCAGATCTAGTCAGACCATTGTACAAATTATGGAAACACCCATTTAGAATGGTTCCTGCA GAAACCAGGCCAGTCTCCAAAGCTCCTGATCTATAAAGTTTCCAACCGATTTTCTGGGGTCCCA GACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTG AGGATCTGGGAATTTATTACTGCTTTCAAGGTTCACATGTTCCATTCACGTTCGGCTCGGGGAC AAAGTTGGAAATAAAA SEQ ID NO: 17 14G11 Antibody Light Chain Variable Region Amino Acid Sequence FR1-CDR1-FR2-CDR2-FR3 -CDR3 -FR4 DVLLTQTPLSLPVSLGDQASISCRSSQTIVQINGNTHLEWFLQKPGQSPKLLIYKVSNRFSGVP DRFSGSGSGTDFTLKISRVEAEDLGIYYCFQGSHVPFTFGSGTKLEIK SEQ ID NO: 18 14G11 Antibody Light Chain CDR1 Amino Acid Sequence RSSQTIVQINGNTHLE SEQ ID NO: 19 14G11 Antibody Light Chain CDR2 Amino Acid Sequence KVSNRFS SEQ ID NO: 20 14G11 Antibody Light Chain CDR3 Amino Acid Sequence FQGSHVPFT SEQ ID NO: 21 Amino Acid Sequence for Epitope 1 Q(Q/L)PYPQ SEQ ID NO: 22 Amino Acid Sequence for Epitope 2 QQPYPQ SEQ ID NO: 23 Amino Acid Sequence for Epitope 3 QLPYPQ SEQ ID NO: 24 Amino Acid Sequence for Epitope 4 Q(Q/L)PYPQ(P/S) SEQ ID NO: 25 Amino Acid Sequence for Epitope 5 QQPYPQP SEQ ID NO: 26 Amino Acid Sequence for Epitope 6 QLPYPQP SEQ ID NO: 27 Amino Acid Sequence for Epitope 7 QQPYPQS SEQ ID NO: 28 Amino Acid Sequence for Epitope 8 QLPYPQS SEQ ID NO: 29 Amino Acid Sequence for Epitope 9 PQ(Q/L)PYPQ SEQ ID NO: 30 Amino Acid Sequence for Epitope 10 PQQPYPQ SEQ ID NO: 31 Amino Acid Sequence for Epitope 11 PQLPYPQ SEQ ID NO: 33 33-mer Amino Acid Sequence Derived from α-2 gliadin LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF