WO2025049981A1 - Variably deamidated gluten polypeptides and antibodies against same - Google Patents

Abstract

Provided are unique antibodies and antigen-binding proteins capable of binding both native and variably deamidated gluten from wheat, barley and rye. Additionally provided are synthetic variably deamidated polypeptides, and methods using same for generating the unique antibodies capable of binding both native and variably deamidated gluten, and cells producing the antibodies. Further provided are methods for using variably deamidated gluten, the synthetic variably deamidated polypeptides, the provided antigen-binding proteins and antibodies and the cells producing same for substantially improved subject/patient monitoring and/or therapeutic management, as well as substantially improved detection of gluten, including variably deamidated gluten in e.g., foods and personal care products, etc.

Description

VARIABLY DEAMIDATED GLUTEN POLYPEPTIDES AND ANTIBODIES AGAINST SAME

FIELD OF THE INVENTION

Aspects of the invention relate generally to monitoring and/or therapeutic management of celiac disease and other gluten sensitivities, as well to the detection of gluten in e.g., foods and personal care products, while more particular aspects relate to synthetic variably deamidated polypeptides, and the use of same in generating unique antibodies capable of binding both native and variably deamidated gluten, where such polypeptides and antibodies provide for substantially improved monitoring and/or therapeutic management, as well as substantially improved detection of gluten, including variably deamidated gluten in e.g., foods and personal care products.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of priority to United States Provisional Patent Application Serial Number 63/536,369, filed 01 September 2023 and entitled VARIABLY DEAMIDATED GLUTEN POLYPEPTIDES AND ANTIBODIES AGAINST SAME, which is incorporated by reference herein in its entirety.

INCORPORATION OF SEQUENCE LISTING

The contents of the xml file named “1111 .xml,” which was created on 20 August 2024, and is 55 KB in size, are hereby incorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

Cereal grains from the botanical tribe Triticeae, including wheat, barley, and rye, constitute an important food source throughout the world (Arendt and Zannini, 2013). Although the nutritional value of these grains is largely derived from carbohydrates, they also contain significant protein content as well (Wieser, 2007). Gluten, the principal protein, is comprised of two fractions; prolamins and glutelins; each consisting of numerous, closely related proteins characterized by a high glutamine (Q) and proline (P) amino acid content and limited solubility in aqueous solution (Wieser, 2007). In particular, the prolamin fractions from wheat, barley, and rye are ordinarily referred to as gliadin, hordein, and secalin respectively. Although gluten is present in all cereal grains, the gluten derived from wheat, barley, and rye is uniquely immunotoxic, capable of inducing symptoms of Celiac Disease (CD) (Briani et al., 2008) as well as other forms of gluten sensitivity (Uhde et al., 2016; Denery-Papini et al., 2012, Sharma et al., 2020).

Among these gluten-related disorders, CD is associated with the highest disease burden. CD is a chronic inflammatory condition classified as a gluten-induced enteropathy that primarily affects the small intestine. CD is estimated to affect roughly 1 % of the worldwide population (Singh et al., 2018). Classic symptoms include diarrhea, constipation, malabsorption, and associated health issues such as anemia, infertility etc., though patients may present non-classically with extra-intestinal manifestations or remain altogether subclinical in their presentation (Fasano, 2005). Left unmitigated, the chronic inflammatory response to dietary gluten may lead to erosion of the intestinal lining and the emergence neoplastic, gluten-responsive T cell clones predisposing CD patients to enteropathy-associated T-cell lymphoma (Smedby et al., 2005).

CD selectively manifests in genetically susceptible individuals possessing HLA- DQ2 and/or DQ8 alleles following consumption of gluten derived from Triticeae grains (Di Sabatino and Corazza, 2009). The pathogenesis of CD is classified as a Type 4 delayed type hyper-sensitivity reaction that involves the generation of CD4+ T helper cells which recognize specific gluten peptides presented within the context of HLA- DQ2/DQ8 molecules on the surface of intestinal dendritic cells in the lamina propria of the small intestine. The recognition of these peptides by CD4+ T helper cells results in the release of inflammatory cytokines such as interferon-gamma which drives local inflammation and villus atrophy in the small intestine, leading to a reduction in the absorptive surface and most of the symptomology (Di Sabatino and Corazza, 2009).

To date, numerous celiaogenic peptides have been described that bind to HLA- DQ2/DQ8, including peptides derived from both the prolamin and glutelin fractions (reviewed in Camarca et al., 2009 and Sharma et al., 2020). Such peptides include, but are not limited to, QQLPQPQQPQQSFPQQQRPF (SEQ ID NO:1 ), (Sjostrom et al., 1998), QQYPSGQGSFQPSQQNPQ (SEQ ID NO:2) (van de Wai et al., 1998), QLQPFPQPQLPY (SEQ ID NO:3) (Arentz-Hansen et al., 2000), LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF (SEQ ID NO:4) (Shan et al., 2002; Qiao et al., 2004), PYPQPQLPY (SEQ ID NO:5) (Arentz-Hansen et al., 2002), FPQQPQQPYPQQP (SEQ ID NO:6) (Arentz-Hanse et al., 2002), QQFSQPQQQFPQPQQ (SEQ ID NO:7) (Arentz-Hansen et al., 2002), LQPQQPFPQQPQQPYPQQPQ (SEQ ID NO:8) (Arentz-Hansen et al., 2002), and GQGIIQPQQPAQLIR (SEQ ID N0:9) (Vader et al., 2002). Of significance, all of these reported peptides are rendered more immunogenic through selective deamidation of glutamine (Q) residues, causing their conversion to glutamic acid (E). Deamidation of the these peptides has been shown to enhance binding to celiaogenic HLA DQ2/DQ8 determinants, thus greatly augmenting recognition by CD4+ T helper cells and the subsequent release of inflammatory cytokines by these cells (Sjostrom et al., 1998; Arentz-Hansen et al., 2000; Qiao et al., 2004; Arentz-Hanse et al., 2002; Vader et al., 2002). Accordingly, deamidated gluten is regarded as significantly more immunotoxic than its native, non-deamidated form.

The effects of deamidation are not limited to immunotoxic T cells; antibodies produced by B cells, including anti-deamidated gliadin— IgA and anti-deamidated gliadin— IgG, typically evolve in the course of CD. Indeed, such antibodies are considered pathognomonic for CD, and are therefore used in the diagnosis of CD (Rashtak et al., 2008). Additionally, deamidated gluten is also known to trigger an acute form of wheat sensitivity characterized as a type I hypersensitivity reaction (Denery- Papini et al., 2012) that involves anti-deamidated gliadin— IgE. In vitro analysis of sera containing these IgE antibodies obtained from patients with allergy to deamidated wheat have demonstrated a direct correlation between degree of gluten deamidation and mast cell activation, thereby establishing deamidated gluten as a highly potent allergen as well (Tranquet et al., 2020).

The deamidation of gluten can occur via different means. Enzymatically, deamidation of gluten is catalyzed via induced glutaminases in food processing or during normal digestive processes via the action of tissue transglutaminase (TGase), an enzyme located within the enterocytes and lamina propria of the small intestine. These enzymes catalyze the ordered and specific conversion of glutamine (Q) to glutamic acid (E) (Molberg et al., 1998; van de Wai, et al., 1998). Deamidation of gluten can occur in an unordered fashion as a consequence of routine food processing involving combined heating and acidification (Mimouni et al., 1994). Furthermore, deamidation of gluten can also occur intentionally in the production of some forms of wheat protein isolate (WPI) (Chen et al., 2021 ) and acid-hydrolyzed wheat protein (HWP) (Tranquet et al., 2017a). The level of deamidation in intentionally deamidated gluten normally ranges from 25-60% deamidation (Tranquet et al., 2017a; Tranquet et al., 2017b). As CD is associated with significant morbidity and there is no cure, patient management is primarily focused on a diet that is devoid of these grains and their products (Hischenhuber et al., 2006; Rubio-Tapia et al., 2013). This dietary stringency has prompted an extensive niche market of gluten-free (GF) food alternatives. However, due to the ubiquitous use of wheat, barley, and rye in the food industry, GF foods are frequently contaminated with gluten (Wieser et al., 2021 ), making avoidance of gluten inherently problematic. Accordingly, to protect against unintentional gluten consumption, numerous countries have adopted regulatory standards that establish gluten limits for foods labeled GF (Ribeiro et al., 2021 ). Enforcement protocols require precise quantification of gluten in foods. The most established techniques for the detection of gluten include ELISA-based assays, PCR, Western Blot, immunochromatography strips, and mass spectrometry. Among these techniques, immunoassays based on monoclonal antibodies directed against specific gluten epitopes are the preferred detection technique. Accordingly, several patents that propose gluten detection procedures based on the ELISA technique currently exist, including WO 2006004394, WO 2006051145, ES 2142720, GB 2207921 , WO 2013045737, and AU 611921. Indeed, several different monoclonal antibodies (mAbs) have been used in commercial gluten detection systems, including R5, A1 , G12, and Skerritt, among which detection assays based on the R5 mAb exhibit superior characteristics. For this reason, R5-based detection assays are regarded as the preferred gluten quantification method by the CODEX Alimentarius and regulatory bodies worldwide ((WHO, 2015; Ribeiro et al., 2021 ).

The R5 mAb targets a-, (3-, and y-gliadin fractions in wheat, specifically at short immunotoxic epitopes including the core peptide, QQPFP (SEQ ID NO: 10) as well as QQQFP (SEQ ID NO:11 ) and LQPFP (SEQ ID NO:12) (Mendez et al., 2005; Kahlenberg et al., 2006), and also including QLPYP (SEQ ID NQ:10), QLPTP (SEQ ID NO:55), QQSFP (SEQ ID NO:56), QQTFP (SEQ ID NO:57), PQPFP (SEQ ID NO:58) and QQPYP (SEQ ID NO:59) (Valdes et al., 2003), which are found plurally within the same gluten molecule and which are present within the afore mentioned immunodominant peptides. However, it has been shown that deamidation of the glutamine residues (Q) significantly ablates the R5 epitopes, thus reducing the effectiveness of R5-based detection methods by an estimated 125 to 600-fold (Kahlenberg et al., 2006; Kanerva et al., 2011 ; Tranquet et al., 2015). Although a monoclonal antibody exists which is capable of recognizing fully deamidated gluten (Tranquet et al., 2015), an antibody with R5-like characteristics that is otherwise capable of recognizing both native and variably deamidated forms of gluten would provide a unique advantage, in that deamidation of gluten is typically incomplete, as previously stated.

BRIEF SUMMARY OF THE DRAWINGS

Figures 1A and B show, according to exemplary non-limiting embodiments of the present invention, indirect ELISA-based analysis of native (NG) and different preparations of deamidated gluten (HWP, DG, and GP). Monoclonal antibodies R5 (Fig. 1A) and 2D4 (Fig. 1 B) were titrated to assess their relative binding profile against the target proteins. Optical density (OD) values at 450 nm, reported as a function of antibody concentration, are graphically represented.

Figure 2 shows, according to exemplary non-limiting embodiments of the present invention, Pepscan analysis of mAb 2D4 using an R5-epitope consensus octapeptide library based on the core QPQQPFPQ.

Figures 3A, 3B and 3C show, according to exemplary non-limiting embodiments of the present invention, SDS-PAGE and comparative Western blot analysis between R5 (art-recognized standard) and 2D4 antibodies, respectively, blotted against prolamins: wheat gliadin (Gli), barley hordein (Hor), rye secalin (Sec), and R5(-) oat avenin (Ave) electrophoretically separated under reducing (denaturing) conditions. MK refers to the molecular weight marker, values in kDa units.

Figures 4A, B and C show, according to exemplary non-limiting embodiments of the present invention, SDS-PAGE and comparative (R5 IgG and 2D4 IgG) Western blot analysis of deamidated gliadins from wheat. Mk refers to molecular weight market, values in kDa units.

Figures 5A and 5B show, according to exemplary non-limiting embodiments of the present invention, indirect ELISA-based analysis of antibodies (R5 (art-recognized standard) and 2D4) against unmodified prolamins and other proteins.

Figures 6A and 6B show, according to exemplary non-limiting embodiments of the present invention, schematics for a Lateral Flow Device for detecting gluten using mAb 2D4. DETAILED DESCRIPTION

Variably Deamidated Polypeptides

Provided are antigen-binding proteins, including monoclonal antibodies raised against VDP-1 (SEQ ID NO:14), a synthetic, variably deamidated R5 tandem epitope (tandem duplicate of the parental epitope LQPQQPFPQQ (SEQ ID NO:13)). A number of antibodies were generated, including mAb 2D4, which exhibited specificity, nearly identical to the art-recognized R5 antibody, for gluten derived from wheat, barley, and rye, but which exhibited substantially improved capability, relative to R5, of detecting deamidated gluten. The 2D4 antibody uniquely detects both native and deamidated (75-100%) gluten. Antibody 2D4 can be applied to substantially improve the scope and sensitivity of testing of food and personal care products, as well as to improve in vitro diagnostic applications to aid in the diagnosis and management of CD and other forms of gluten sensitivity.

Additionally provided are 20-amino acid variably deamidated peptide sequences corresponding to LZPZZPFPZZLZPZZPFPZZ (SEQ ID NO: 14; wherein “Z” is Q or E) alternately referred to herein as ”VDP-1”. In particular aspects, VDP-1 is a synthetic 20-aa peptide consisting of a duplicated linear core epitope LQPQQPFPQQ (SEQ ID NO: 13) of gluten that is variably and randomly substituted with glutamate at the glutamine residue positions. This core epitope occurs within a known clinically significant immunodominant native gluten epitope LQPQQPFPQQPQQPYPQQPQ (SEQ ID NO:8), which has been implicated in the pathogenesis of CD, and shown to exert increased immunotoxicity when deamidated at specified glutamine residues (Arentz-Hansen et al., 2002; Camarca et al., 2009). The core peptide sequence LQPQQPFPQQ (SEQ ID NO: 13) also occurs in a highly reiterated manner in gluten derived from wheat, barley, and rye, as revealed by blastp analysis of non-redundant protein sequences (https: //blast, ncbi. nlm. nih. gov/Blast. cgi?PAGE = Proteins).

VDP-1 was synthesized via random substitution of glutamine with glutamic acid and comprise a stochastic mixture of different peptide species, including LQPQQPFPQQLQPQQPFPQQ (SEQ ID NO:15), which is a duplicate of the core peptide LQPQQPFPQQ (SEQ ID NO: 13), along with random amino acid sequence variations thereof. Specifically, these deamidation variations were introduced, during peptide synthesis, by random substitution of glutamine (Q) with glutamic acid (E) at the glutamine residue positions, to achieve the variably deamidated sequence LZPZZPFPZZLZPZZPFPZZ (SEQ ID NO: 14; wherein “Z” is Q or E). According to aspects of the invention, this synthetic approach sufficiently mimics the gluten epitope deamidation variants resulting from tissue transglutaminase activity in the intestine and/or from the effects of food processing and chemical production methods. Such variations can cause significant changes in the physical and/or biological characteristics of the peptide, altering e.g., solubility in aqueous solution, and/or antigenicity and/or immunotoxicity. Also within the scope of the present invention are polypeptides containing an amino acid sequences comprising reiterations of LZPZZPFPZZLZPZZPFPZZ (SEQ ID NO: 14).

Additional aspects provide isolated 20-mer polypeptides VDP-2, VDP-3 and VDP-4 having the sequences LZPZQPFPZZLZPZZPFPZZ (SEQ ID NO:46), LZPZZPFPZZLZPZQPFPZZ (SEQ ID NO:47), LZPZQPFPZZLZPZQPFPZZ (SEQ ID NO:48), respectively, wherein relative to VDP-1 (SEQ ID NO: 14), the residues at positions 5 and/or 15 are not variably deamidated — reflecting the Pepscan analysis of the R5-epitope consensus octapeptide QPQQPFPQ (SEQ ID NO: 16). According to further aspects, therefore, the VDP-2, (SEQ ID NO:46), VDP-3 (SEQ ID NO:47) and/or VDP-4 (SEQ ID NO:48) polypeptides, or concatenates or mixed concatenates thereof, may be used as an immunogen, in analogy to VDP-1 (SEQ ID NO: 14), for generating, and/or screening for antibodies that recognize variably deamidated gluten.

The disclosed polypeptides (e.g., VDP-1 , VDP-2, VDP-3, VDP-4, and concatenates and mixed concatenates thereof) may comprise modifications at either the N terminus or C terminus, or both. Such modifications provide for use of the polypeptides in, for example, biosensor applications to detect antibodies, e.g., in patients with suspected or known gluten sensitivities, in antigen applications to assess B cell and T cell responses in such patients, or to facilitate raising antibodies for detecting variably deamidated gluten in various matrices including but not limited to food, personal care products, etc., or in human biological samples. Exemplary terminal modifications include, but are not limited to the addition of biotin, an enzyme such as HRP or ALKP, a fluorogenic substance, a radioactive substance, a chemiluminescent substance, an electrogenerated chemiluminescent substance, a metallic nanoparticle or any chemical modification for enhancing the immunogenicity or bioavailability of the peptide(s) of the invention, including the addition of carrier proteins, arrangement as MAPS, or PEGylation. Antigen-binding Proteins and Antibodies

Additionally provided are antigen-binding proteins (e.g., antibodies) that bind to variably deamidated gluten for use in gluten detection systems. Methods for generating such suitable antibodies using the disclosed polypeptides (e.g., VDP-1 , VDP-2, VDP-3, VDP-4, and concatenates and mixed concatenates thereof) are also provided. Particular embodiments provide antibodies, that bind to variably deamidated gluten, with hypervariable region sequences that make direct contact with variably deamidated gluten, and define its antigen specificity. The sequences and CDR’s of the 2D4 antibody, for example, are characterized and summarized in working Example 4 herein below.

Methods for generating antibodies that bind to variably deamidated gluten comprise, for example, immunizing animals with a variably deamidated immunogenic polypeptide (e.g., VDP-1 , VDP-2, VDP-3, VDP-4, and concatenates and mixed concatenates thereof) with subsequent hybridoma generation and screening, and purification of specific antibodies generated against it. Such a representative method, used to generate mAb 2D4, is disclosed herein below in working Example 1. Alternatively, EBV-transformed B lymphoblast cell lines can be generated using peripheral blood mononuclear cells isolated from patients with celiac disease or other forms of gluten sensitivity.

The provided antibodies that bind to variably deamidated gluten may be polyclonal (typically including different antibodies directed against different determinants or epitopes) or monoclonal (involving a single antibody directed against a single determinant or epitope). The provided antibodies may be altered biochemically, by genetic and/or recombinant manipulation, or synthetically. A resulting altered antibody may lack, in its totality or parts, portions that are not required for the recognition of its variably deamidated gluten target, and such portions may be replaced or substituted by other portions that provide further advantageous properties to the altered antibody. Accordingly, antibodies of the invention not only include intact immunoglobulins, but also include immunologically active portions or molecules derived from immunoglobulins, e.g., molecules that contain an antigen binding site which specifically contacts variably deamidated gluten. Representative examples of portions of immunologically active molecules from immunoglobulins include the hypervariable regions, also known as Complementarity-determining regions (CDRs), F(ab) fragments, and F(ab')2 fragments, which can be generated, for example, by treating the antibody with a protease or by genetic engineering techniques, as recognized in the art.

The provided antibodies exhibit specificity for variably deamidated gluten, which has substantial utility in many applications, for example, as in the detection and/or quantification of variably deamidated gluten in food, personal care products, or biological samples isolated from an individual in view of making in vitro diagnosis and/or monitoring of gluten-related disorders. Significantly, the provided antibodies are capable of detecting variably deamidated gluten peptides, including peptides which are extensively deamidated as well as peptides which are not deamidated. This promiscuous ability to detect a poly peptide target that is variably deamidated is a unique and defining feature of the provided antibodies of the invention.

Antibody Producing Cells

Additional aspects provide cells that express the respective provided antibodies. The provided cells producing the provided antibodies are preferably a B lymphocyte, an EBV-transformed B lymphoblast, or a hybridoma, the later consisting of a hybrid cell line generated by fusing a B lymphocyte producing a respective provided antibody with a myeloma cell line (cancerous B lymphocyte) to form an immortalized cell line capable of producing the respective provided antibody ad infinitum. A monoclonal antibody can be produced from a respective producing cell line using various methods, including but not limited to in vivo production in ascetic fluid, or in vitro production in tissue culture medium. Such cell lines can include mouse hybridoma cell lines or human EBV- transformed B lymphoblast cell lines derived from peripheral-blood mononuclear cells obtained from patients with CD or other gluten sensitivities.

Further aspects provide methods using the provided antibodies and/or respective cells producing same, for the detection, enrichment and/or quantification of variably deamidated gluten. For example, the antibody of the invention can be used to detect, enrich, and/or quantify variably deamidated gluten in food, personal care products, or human biological samples isolated from patients with celiac disease or other gluten sensitivities. Such representative applications can be used, for example, to assure gluten-free status of foods and personal care products intended for consumption or use by individuals with celiac disease or other gluten sensitivities. Representative applications also include those that can be used to measure variably deamidated gluten in biological samples, such as urine, feces, or blood products, to assess dietary compliance in patients with CD or to monitor emerging therapeutic interventions in patients with CD. Preferred embodiments include applications in the detection and/or quantification of variably deamidated gluten in foods, personal care products, and human biological samples.

Compositions

Also provided are compositions comprising: variably deamidated gluten and/or the disclosed variably deamidated gluten polypeptides (e.g., VDP-1 , VDP-2, VDP-3, VDP-4, and concatenates and mixed concatenates thereof); the provided antibodies (e.g., 2D4, and/or antigen-binding portions thereof); and/or the respective antibody producing cells. The compositions may be formulated for administration using a variety of art-recognized techniques. Examples of such formulations include, but are not limited to any solid (tablets, pills, capsules, granules, etc.) or liquid (solutions, suspensions or emulsions) formulated for oral, rectal, topical, percutaneous, or parenteral administration. The provided compositions may also be formulated as or in liposomes or as nanospheres, sustained release formulations, or any other conventional release system.

Variably deamidated gluten polypeptides can be produced naturally in the gut from ingested gluten though the action of tissue transglutaminase, which deamidation is immunologically consequential in the context of gluten-related sensitivities. The provided variably deamidated gluten and/or the variably deamidated polypeptides (e.g., VDP-1 , VDP-2, VDP-3, VDP-4, and concatenates and mixed concatenates thereof) and compositions comprising same, therefore, have utility in the diagnosis, management, prevention and/or treatment of celiac disease and other gluten sensitivities. Possible medical applications additionally encompass uses of the provided cells, and/or antibodies, or compositions comprising same, to aid in the diagnosis, management, prevention and/or treatment of celiac disease and other gluten sensitivities.

The provided variably deamidated polypeptides, cells, antibodies, and/or compositions comprising same may, for example, be introduced percutaneously, to assess for a Type 4, delayed type hypersensitivity reaction towards the peptide of the invention to aid in the diagnosis of CD. The provided polypeptides may additionally be used to assess for a type I immediate hypersensitivity reaction towards variably deamidated gluten polypeptides to aid in the diagnosis of allergy directed towards deamidated gluten. In yet further examples, the provided antibodies that bind to variably deamidated gluten, and/or mitogen-activated B cells prepared from the blood of patients with gluten sensitivities, may be used to monitor response to therapy. The provided variably deamidated gluten and/or the variably deamidated polypeptides (e.g., VDP-1 , VDP-2, VDP-3, VDP-4, and concatenates and mixed concatenates thereof) are also useful as a therapeutic agents, for example, as a vaccine or immunogen to desensitize gluten-allergic patients or to clonally delete pathogenic T cells in patients with CD so as to induce immunological tolerance. Further aspects, therefore, encompass the use of the provided variably deamidated gluten polypeptides, cells, antibodies, and/or compositions comprising same, in the diagnosis, management, prevention and/or treatment of celiac disease and other gluten sensitivities.

Kits

Further aspects provide kits or combinations comprising: variably deamidated gluten and/or the provided variably deamidated polypeptides (e.g., VDP-1 , VDP-2, VDP-3, VDP-4, and concatenates and mixed concatenates thereof); the provided antigen-binding proteins (e.g., 2D4 antibody and/or antigen-binding portions thereof, etc.); the provided cells (e.g., hybridomas) producing the provided antibodies; and combinations thereof. The provided kits or combinations may comprise, without limitation, conjugated or free antibodies, peptides, buffers, conjugated secondary antibodies, conjugated streptavidin, protein or peptide standards, agents for pollution prevention, marker compounds, as for example, but not limited to, fluorochromes, chromogens, etc. The provided kits or combinations, moreover, may include containers and/or solutions for implementation and optimization. The provided kits or combinations may contain other or additional proteins or peptides that serve as positive and negative controls. The provided kits or combinations have utility, for example, to monitor disease progression in individuals with CD or other gluten sensitivities. The provided kits or combinations have utility, for example, to detect, enrich, and/or quantify variably deamidated gluten polypeptides, anti-gluten antibodies, and/or cells producing same.

In preferred embodiments, the provided kits or combinations have utility for the testing of food, personal care products, or human biological samples (e.g., to aid in the diagnosis and/or monitoring of celiac disease or other gluten sensitivities). For example, the provided kits combinations may be used to assess foods or personal care products for the presence and/or to quantify gluten content so as to ensure the suitability of commodities expressively intended for patients with celiac disease or other gluten sensitivities. In other preferred embodiments, the provided kits or combinations may be used on human biological samples, such as plasma, serum, blood, immune cells, urine, or feces, to detect, enrich, and/or quantify variably deamidated gluten polypeptides, anti-gluten antibodies, and/or cells producing same, to aid in the in vitro diagnosis and/or monitoring of celiac disease or other gluten sensitivities.

Exemplary provided kits and combinations include, but are not limited to, those for Western blot (as described herein below in working Example 5), immunoprecipitation, immune-magnetic separation, affinity-chromatography, protein arrays, immunochromatographic assays (e.g., lateral flow devices), flow-through assays, immunohistochemistry-based assays, immune-fluorescent-based assays, ELISA (e.g., direct, indirect, competitive or sandwich), or ELISpot. Kits or combinations comprising the provided antibodies (e.g., 2D4 and antigen-binding portions thereof, etc.) may be used, for example, to detect, enrich, or quantify the variably deamidated gluten polypeptides present in human biological samples isolated from individuals with celiac disease or other gluten sensitivities. Likewise, kits or combinations comprising the provided antibodies (e.g., 2D4 and/or antigen-binding portions thereof, etc.) can be used to detect, enrich, or quantify the peptide present in food or personal care products. An advantage of using a kit that specifically detects variably deamidated gluten polypeptides, rather than only native gluten polypeptides peptides, is that variably deamidated gluten polypeptides are naturally occurring in the body through the action of tissue transglutaminase present within the small intestine, which converts native gluten to a variably deamidated form. Additionally, variably deamidated gluten polypeptides are present in cereal products derived from barley, rye, or wheat that have undergone enzymatic or chemical processing. Furthermore, the ability of the provided antibodies to bind to the random variability in deamidation present within variably deamidated gluten polypeptides enables cost-effective assessment and treatment broadly across the human population regardless of host immunogenetics.

In the provided kits and combinations, variably deamidated gluten and/or the variably deamidated polypeptides (e.g., VDP-1 , VDP-2, VDP-3, VDP-4, and concatenates and mixed concatenates thereof), the antibodies (e.g., 2D4, and/or antigen-binding portions thereof, etc.), and/or the respective antibody-producing cells (e.g., hybridomas, etc.) may be labeled or immobilized. Preferably, they are marked with a label selected from, but not limited to, the group consisting of: a radioisotope, a fluorescent or luminescent label, an antibody, an antibody fragment, an affinity tag, an enzyme or an enzymatic substrate. More preferably, the variably deamidated gluten and/or the variably deamidated polypeptides (e.g., VDP-1 , VDP-2, VDP-3, VDP-4, and concatenates and mixed concatenates thereof), the antibodies (e.g., 2D4, and/or antigen-binding portions thereof, etc.), and/or the respective antibody-producing cells (e.g., hybridomas, etc.) are absorbed or immobilized in the kits of the invention. The term “immobilized” as used herein, refers to the provided variably deamidated polypeptides, the provided antibodies or the provided cells producing same, which components may be attached to a support without losing their respective activities/functionality. The support may, for example, be the surface of a matrix (e.g., a nylon or nitrocellulose matrix), a microtiter plate (for example a 96-well plate) or similar plastic support, a flow-through device membrane, or beads (spheres, including but not limited to spheres of agarose, microspheres composed of magnetic matrices, or nanoparticles composed of metals). In particular, the immobilization of the provided antibodies (e.g., 2D4, and/or antigen-binding portions thereof, etc.) to agarose or magnetic microspheres may facilitate enrichment of variably deamidated gluten polypeptides from a matrix such as a food or biological sample.

Preferred exemplary kits and methods take the form of, or provide for a sandwich enzyme-linked immunoassay, or ELISA, based, e.g., on the use of a pair of the provided antibodies (e.g., 2D4, and/or antigen-binding portions thereof, etc.) having affinity for a plurality of epitopes present within gluten (e.g., gluten, and/or variably deamidated gluten and/or the variably deamidated polypeptides (e.g., VDP-1 , VDP-2, VDP-3, VDP-4, and concatenates and mixed concatenates thereof) that are sufficiently far enough apart to allow for the paired interaction of the two antibody molecules with the same target molecule simultaneously. For example, one member of the antibody pair is bound to a solid support (e.g., a plastic well, membrane, etc.) and the second member of the antibody pair is used as a detector, conjugated, for example, with an enzyme that catalyzes a chromogenic reaction, or a fluorochrome (in fluorometric techniques). The test sample is incubated with these reagents and the ensuing color intensity or emitted fluorescence is measured/observed during, and/or at the end of the process, being directly or otherwise proportional to the amount of peptide present in the sample within a defined range of quantification. Working Example 7, herein below, describes how the exemplary 2D4 antibody was used in sandwich ELISA methods and kits. In additional exemplary aspects for detection and quantification of variably deamidated gluten polypeptides, the kits and methods take the form of, or provide for a competitive immunoassay format in which the variably deamidated gluten, and/or variably deamidated synthetic polypeptides (e.g., VDP-1 , VDP-2, VDP-3, VDP-4, and concatenates and mixed concatenates thereof) is absorbed to a solid phase and then exposed to: a provided antibody (e.g., 2D4, and/or antigen-binding portions thereof, etc.), preferably conjugated with a chromogenic enzyme or fluorochrome; and the test sample. Alternatively, the competitive assay may be configured such that the provided antibody (e.g., 2D4, and/or antigen-binding portions thereof, etc.) is absorbed onto the solid phase and then incubated with sample and an antigen (e.g., variably deamidated gluten, and/or variably deamidated synthetic polypeptides (e.g., VDP-1 , VDP-2, VDP-3, VDP-4, and concatenates and mixed concatenates thereof) conjugated to a chromogenic enzyme or fluorochrome. In either case, the amount of peptide detected is inversely proportional to the color evolved or the fluorescence emitted. The competitive technique has particular utility in the detection of hydrolyzed gluten residues, which typically contains deamidated gluten epitopes, and may be present as consequence of certain food processing techniques or enzymatic processes.

Other exemplary kits and methods for detection and quantification of variably deamidated gluten polypeptides take the form of, or provide for an indirect ELISA in which variably deamidated gluten, and/or variably deamidated synthetic polypeptides (e.g., VDP-1 , VDP-2, VDP-3, VDP-4, and concatenates and mixed concatenates thereof) is/are absorbed to a solid phase, and then exposed to an antibody (e.g., the provided antibody (e.g., 2D4, and/or antigen-binding portions thereof, etc.) or an antiserum containing an anti-gluten antibody, which is subsequently serially diluted and subsequently probed using a secondary antibody that is conjugated to a suitable label. In this way, the kit may also be used to measure antibodies in serum isolated from patients with celiac disease or other forms of gluten sensitivity by measuring antigenspecific IgG, IgA, or IgE titers to aid in the diagnosis and monitoring of such diseases. Working Example 6, herein below, describes how the exemplary 2D4 antibody was used in indirect ELISA methods and kits.

Another exemplary kit and method for detection and/or quantification of variably deamidated gluten polypeptides takes the form of, or provides for an immunochromatographic technique, wherein the provided antibody (e.g., 2D4, and/or antigen-binding portions thereof, etc.) is adsorbed on a membrane, or, bound to a colored nanoparticle such as gold, latex, or polystyrene that is used as a detection substance. This technique allows for the rapid qualitative determination of gluten in a sample. Alternatively, the immunochromatographic technique can be modified so that the antigen (e.g., variably deamidated gluten, and/or variably deamidated synthetic polypeptides (e.g., VDP-1 , VDP-2, VDP-3, VDP-4, and concatenates and mixed concatenates thereof) is instead adsorbed on the membrane and probed using a detector consisting of the provided antibody (e.g., 2D4, and/or antigen-binding portions thereof, etc.) conjugated to, e.g., a detectable nanoparticle such as gold particles, in this way functioning as a competitive assay that enables detection of highly hydrolyzed gluten samples. Working Examples 8 and 9, herein below, describes how the exemplary the 2D4 antibody was used in Immunochromatographic/lateral flow methods and kits.

Additional preferred kits and methods for detection take the form of, or provide for ELISpot techniques. ELISpot can be used to quantify CD4+T cell responsiveness against variably deamidated gluten, and/or variably deamidated synthetic polypeptides (e.g., VDP-1 , VDP-2, VDP-3, VDP-4, and concatenates and mixed concatenates thereof). Patient peripheral blood mononuclear cells are incubated with variably deamidated synthetic polypeptides (e.g., VDP-1 , VDP-2, VDP-3, VDP-4, and concatenates and mixed concatenates thereof) for a period of time and then added to wells that contain a capture antibody specific for an inflammatory cytokine, for example, interferon-gamma for a period of time. The mixture is then removed and the wells are probed for the cytokine using a second chromogen-conjugated antibody that is likewise specific for the cytokine under scrutiny. The spots that are evolved in the well after the addition of the chromogen indicate individual T cells that are reactive against variably deamidated synthetic polypeptides (e.g., VDP-1 , VDP-2, VDP-3, VDP-4, and concatenates and mixed concatenates thereof), and can therefore be used to enumerate celiaogenic T cells present in the peripheral blood of patients. ELISpot can, in this exemplary way, be a useful tool to monitor the disease, including responsiveness to treatment without necessitating invasive procedures such as endoscopy-guided biopsy.

Throughout the description and claims the word “comprise” and its variants are not intended to exclude other technical features, additives, components or steps. To those skilled in the art, other objects, advantages and features of the invention will become apparent in part from the specification and in part from the practice of the invention. The following examples and figures are provided by way of illustration, and are not intended to be limiting of the present invention.

EXAMPLE 1

(Materials and Methods)

Production of monoclonal antibodies. BALB/c female mice (Charles River Laboratories, Wilmington, MA, USA) were immunized 3-5 times with VDP-1 LZPZZPFPZZLZPZZPFPZZ (SEQ ID NO: 14; wherein “Z” is Q or E) (50 to 100 pg mixed with either complete Freund’s adjuvant), a synthetic, variably deamidated tandem linear epitope based on the R5 footprint generated using solid-phase peptide synthesis technology. To generate hybridomas, splenocytes were fused to SP2/O-Ag14 myeloma cells using PEG 1500 (Roche, San Francisco, CA, USA) and cultured in Dulbecco's Modified Eagle Medium DMEM supplemented with HAT medium (ATCC, Manassas, VA, USA) and 10% fetal bovine serum (GE Healthcare Life Sciences HyClone Laboratories, Logan, UT, USA). Resulting colonies were manually selected, clonally expanded, and screened by ELISA. lgG+ clones (IsoStrip™ Kit (Sigma-Aldrich, St. Louis, MO, USA)) that remained stable and exhibited high reactivity against gliadin, hordein, and secalin, such as clone 2D4, were further studied to identify suitable candidates.

Purification of monoclonal antibody. Clone 2D4 cells were expanded in vitro under stepwise serum reduction conditions. Cultures were maintained until cells were exhausted and no viable cells remained. Supernatant was harvested, spun down, and passed through a 0.2 pm filter. Purification was performed using a protein G column on a GE AKTA Prime System (Cytiva Life Sciences, Marlborough, MA, USA) and eluted with 0.05 M glycine buffer, pH 2.8. The eluted fractions were dialyzed overnight against phosphate-buffered saline (PBS). The R5 antibody used in the comparative analyses was kindly provided under a licensing agreement with the Spanish National Research Council (CSIS).

Antibody sequencing and CDR definition. Total RNA from hybridoma cell line 2D4 was extracted using Trizol (Thermo Fisher Scientific) and RNasey (Qiagen) methods. NEB Template Switching RT Enzyme Mix was used for first strand cDNA synthesis. During second strand synthesis, isoform-specific 5’ primers were used for heavy chain amplification, and diverse kapa and lambda chain primers were used for light chain amplification. During the second strand synthesis and subsequent PCR, only G1 isoform -specific primers yielded a PCR product for heavy chain sequencing, and only the kappa gene-specific primer yielded a positive product for light chain sequencing. PCR products were then amplified with Illumina Nextera adaptors to create an amplicon library. A summary of the relevant primers is provided in Table 8 below:

Table 8. Primer summary.

Illumina MiSeq NGS sequencing was done using established SOPs. The RNA Spades algorithm (https: //cab.spbu.ru /software Zrnaspades /) was employed for read alignment of Illumina RNA-Seq data. Full-length sequences were identified and matched to heavy and light chain types using the IMGT webserver (https: //www. imgt.org /IMGT_vquest/input), incorporating IMGT's CDR information. Antibody sequences and CDR are described in Example 4 below.

Preparation of prolamins. Flours from American wheat (King Arthur Baking Company, Norwich, VT, USA), barley (Food to Live, Brooklyn, NY, USA), and rye (Bob’s Red Mill, Milwaukie, OR, USA) as well as non-milled rice, millet, corn, and soy were locally purchased at a supermarket. Native gliadin used as a reference material was obtained from Raisio Grain Starch, Ltd (Finland). For native gluten, Osborne fractionation was undertaken to isolate the prolamin fractions (gliadin, hordein, and secalin); first, by removing globulins and albumins from the flour by repeated extractions using a 0.5 M NaCI followed by an extraction of the solids using 60% ethanol (v/v) solution. Protein concentration of unmodified prolamins was determined by combustion analysis with a Dumas analyzer (FP-328 combustion instrument Leco, Kirchheim, Germany), and calculated by multiplying the N-content by a co-efficient of 5.7. Deamidated gluten samples used as references samples are previously described and included enzymatically deamidated samples prepared using protein glutaminase (PG) for 1 h, 5h and 30h resulting in deamidation levels of 20%, 60% and 72% respectively (Tranquet et al., 2017b). Hydrolyzed Wheat Proteins (HWP) 1 , 2, 3, and Glupearl-19S (GP19S) were industrially prepared using a combined acid and heat treatment with reported deamidation levels of 25-30%, 60%, 50-60%, and 50-60%, respectively (Tranquet et al., 2017a; Tranquet et al., 2017b).

Indirect and Sandwich ELISA. For indirect ELISA: cereal prolamins, soy protein, and deamidated gliadin samples were dissolved in 60% ethanol (v/v) and then dissolved in carbonate buffer, pH 9.8 to 5 pg/mL for unmodified samples and 1 pg/mL for deamidated samples. All targets were plated in 100 pL aliquots into 96-well microtiter plates, then fixed and blocked overnight. To assess affinity, unlabeled antibodies were initially diluted down to 0.1 mg/mL and then serially diluted in 1 % BSA solution. 100 pL of each dilution was aliquoted into wells and incubated for 10 mins at room temperature. Plates were washed with PBST and incubated with 100 pL goat anti-mouse IgG HRP conjugate (1/2000; Sigma) diluted in commercially available HRP conjugate diluent for 10 minutes at room temperature. Plates were washed repeatedly with PBST and resolved using 100 pL of 3,3',5,5'-Tetramethylbenzidine (TMB) solution for 5 mins.

For sandwich ELISA, unlabeled 2D4 was plate bound (1.4 pg/mL) into 96-well microtiter plates using PBS buffer, pH 7.4 and subsequently blocked overnight. Prolamin Working Group (PWG) Gliadin standards and deamidated gliadin samples were diluted 20-fold in 1% BSA solution and then 100 pL was added to each well in duplicate. These were incubated for 20 minutes at room temperature. Wells were washed 5X with PBST and then incubated with 100 pL 2D4 HRP-conjugate, diluted in commercially available HRP stabilizer buffer, for a further 20 minutes. After a second wash, the plates were resolved using 100 pL of TMB solution for 10 mins. The TMB chromogenic reactions were terminated by the addition of 100 pL of 1 M phosphoric acid and the OD values were measured using a Thermo Scientific MultiSkan plate reader set with a bichromatic 450/650 filter. The data were fitted to a modified Michelis- Menton equation and analyzed using GraphPad PRISM (GraphPad Software, San Diego, CA). ELISAs were repeated independently a minimum of two times and the results presented are an average of the replicates.

Epitope mapping with solid-phase synthetic peptides (Pepscan). Pepscan analysis was performed as previously described (Tranquet et al., 2017b). In brief, an octapeptide library was prepared by automated spot synthesis and covalently attached to a cellulose membrane (Abimed, Langenfeld, Germany). The printed membrane was washed and then blocked with 2.5% (w/v) non-fat skim milk (NFSM) powder and 5% (w/v) sucrose in Tris-buffered saline with 0.05% Tween-20 (TBST) for 1 h. The membranes were incubated overnight with 2D4 (1 pg/mL). After washing, the membranes were incubated with HRP-conjugated anti-mouse IgG (170-6516, Bio-Rad Laboratories, Hercules, CA, USA) diluted 1/50,000 for 1 h and then washed again. The membranes were treated with chemiluminescent substrate (#K12042, Advansta San Jose, CA, USA) and the results were captured using a CCD camera (Luminescent Image Analyzer LAS 3000; Fujifilm, Tokyo, Japan).

Electrophoresis and western blot analysis. For electrophoretic analysis, prolamins (10 pg) were denatured using SDS and beta-mercaptoethanol and resolved using a 10% acrylamide gel run with SDS-glycine buffer at 200 V using a Mini Protean III Cell Apparatus (Bio-Rad Laboratories, Hercules, CA, USA). For western blotting, 0.75 pg of prolamin protein samples were loaded and operated as described above. Gels were rinsed and then transferred to a nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA, USA) using a Trans-Blot® Turbo Transfer System (BioRad Laboratories, Hercules, CA, USA). Membranes were blocked using 1 % NFSM powder dissolved in TBST and then incubated with 560 ng/mL of 2D4 or R5 IgG in TBST-1 % NFSM for 1 h. Blots were washed and then incubated with goat anti-mouse IgG-HRP conjugate (1 :20,000 in TBST) for 1 h, washed repeatedly, then resolved using Pierce ™ 1-Step Ultra TMB Blotting Solution (Thermo Scientific, Rockford, IL, USA). The chromogenic reaction was stopped by addition of water. Gel and membranes were imaged using Gel Doc ^TM EZ Imager (Bio-Rad Laboratories, Hercules, CA, USA).

EXAMPLE 2

(The VDP-1 peptide (SEQ ID NO: 14) was used as an immunogen to generate hybridomas and antibodies)

VDP-1 peptide (LZPZZPFPZZLZPZZPFPZZ (SEQ ID NO: 14)) was used, as described in Example 1 , as an effective immunogen in mice to generate hybridomas that express antibodies with the ability to recognize both native and deamidated forms of gluten. Resulting hybridoma clones were screened by indirect ELISA (as described in Example 1) against both native gliadin (prolamin from wheat) and deamidated gliadin.

Figures 1A and 1 B show the results for exemplary hybridoma clone 2D4, comparing the performance of its monoclonal antibody 2D4 against the art-recognized gold standard mAb R5. Specifically, Figures 1A and B show, according to exemplary non-limiting embodiments of the present invention, indirect ELISA-based analysis of native and deamidated gluten. Target protein (1 pg/mL) was immobilized on the well surface. Target proteins included a panel of deamidated prolamins and native gliadin (NG) as a control, including HWP1 , HWP2, HWP3, DG, GP19S, PG-1 h, PG-5h, and PG-30h. See Table 1 below for a summary description of each target. Monoclonal antibodies R5 (Fig. 1A) and 2D4 (Fig. 1 B) were titrated to assess their relative binding profile against the target proteins. Optical density (OD) values at 450 nm, reported as a function of antibody concentration, are graphically represented.

The samples used in this analysis were clinically significant, each having been documented in triggering adverse allergic reactions in humans as previously reported (Tranquet et al., 2015; Tranquet et al., 2017a; Tranquet et al., 2017b) (see Methods section). The half-binding concentration values of 2D4 and R5 for the different targets are presented in Table 1 .

Table 1. Comparative half-binding concentration values obtained by Indirect ELISA*

*Values obtained using plate-bound antigen ** Not determinable

In summary, although both antibodies exhibited similar affinity for native gliadin (NG), R5 demonstrated significantly reduced affinity for deamidated gliadin samples compared with mAb 2D4, in a manner that was inversely proportional to the degree of deamidation. This reduction in affinity was observed whether the deamidation was achieved chemically or enzymatically. Specifically, 2D4 demonstrated half-binding concentrations that were between 1.8 times and 50 times lower than R5, indicating superior affinity. Importantly, for samples that were very heavily deamidated, including the hydrolyzed wheat protein 3 (HWP3) sample, which was 50-60% deamidated, and a sample enzymatically prepared using 30h treatment with glutaminase (PG-30h), which was 72% deamidated, the R5 half-binding affinities could not be determined, whereas the 2D4 affinities were still calculable for these more highly processed samples. This example demonstrates that the peptide of the invention is capable of generating a cell of the invention that is in turn capable of producing an antibody of the invention that can recognize both native and deamidated forms of gluten.

EXAMPLE 3

(The 2D4 antibody was shown to promiscuously recognize variable epitopes contained within the R5-epitope consensus octa peptide QPQQPFPQ SEQ ID NO: 16)

One of the key features of the VDP-1 immunogenic peptide (SEQ ID NO: 14) is that it is variable in composition. According to aspects of the invention, employing VDP- 1 as an immunogen, would result in the ensuing antibodies being able to exhibit some degree of promiscuity, in detecting variably deamidated epitopes. To confirm this, exemplary antibody 2D4, was subjected to epitope mapping using Pepscan analysis (Figure 2)

Specifically, Figure 2 shows, according to exemplary non-limiting embodiments of the present invention, Pepscan analysis of exemplary mAb 2D4 using an Octapeptide library. Specifically, Pepscan analysis of 2D4 was performed using immobilized R5-epitope consensus octapeptide QPQQPFPQ (SEQ ID NO: 16) and its substitution permutations using glutamic (deamidation; rows 10-24) acid or alanine (control; rows 1-9). Membranes were immunoblotted with 2D4 mAb produced in vivo from ascites (#20-245A) and in vitro from tissue culture (#21-148). Secondary antibody alone (2^nd Ab only) was run as a control.

In brief, an octapeptide library was generated using the parental R5-epitope consensus octapeptide QPQQPFPQ (SEQ ID NO: 16), the distilled core of the peptide of the invention, and analogs thereof based on sequential substitutions with either alanine (rows 1-9) or glutamic acid (rows 10-24. The peptides were covalently attached to a membrane and then immunoblotted with mAb 2D4. Rows 1 to 9 represent peptide variants in which each amino acid was sequentially altered to alanine (A). The data indicates that proline (P) at positions 2 and 5 as well as glutamine (Q) at position 4 and phenylalanine (F) at position 6 are essential residues for 2D4 binding, thus identifying a consensus sequence of XPXQPFX. Rows 10-24 represent peptide variants in which each glutamine residue was sequentially replaced by glutamic acid, essentially mimicking deamidation. Deamidation at positions 1 and 3 did not significantly affect 2D4 binding. However, if both the glutamines in positions 4 and 8 were replaced by glutamic acid (rows 19, 21 , 23, 24), 2D4 binding was significantly reduced. Thus, exemplary mAb 2D4 not only recognizes the native epitope QPQQPFPQ (SEQ ID NO: 16), but also partially deamidated epitopes (up to 75% deamidated). The inherent promiscuity of exemplary mAb 2D4, generated using the immunogen VDP-1 (SEQ ID NO: 14), permits binding to variably deamidated gluten epitopes, and thus represents a key aspect of the invention. By comparison, epitope recognition by typical antibodies is specific and not promiscuous.

Additional aspects provide isolated 20-mer polypeptides VDP-2, VDP-3 and VDP-4 having the sequences LZPZQPFPZZLZPZZPFPZZ (SEQ ID NO:46), LZPZZPFPZZLZPZQPFPZZ (SEQ ID NO:47), LZPZQPFPZZLZPZQPFPZZ (SEQ ID NO:48), respectively, wherein relative to VDP-1 (SEQ ID NO: 14), the residues at positions 5 and/or 15 are not variably deamidated — reflecting the Pepscan analysis of the R5-epitope consensus octapeptide QPQQPFPQ (SEQ ID NO: 16). According to further aspects, therefore, the VDP-2, (SEQ ID NO:46), VDP-3 (SEQ ID NO:47) and/or VDP-4 (SEQ ID NO:48) polypeptides, or concatenates or mixed concatenates thereof, may be used as an immunogen, in analogy to VDP-1 (SEQ ID NO: 14), for generating, and/or screening for antibodies that recognize variably deamidated gluten.

The promiscuity of exemplary mAb 2D4 is significant in that while deamidation of gluten, when it occurs, is typically <70, detection and/or quantification of such partially deamidated gluten is nonetheless inherently problematic for traditional detection methods based on conventional antibodies (e.g., R5) that solely recognize native gluten peptides or fully deamidated gluten peptides. The promiscuity of exemplary mAb 2D4 is also surprising, since deamidation of Q to D results in a chare reversal.

In summary, detection and/or quantification methods based on antibodies (e.g., exemplary mAb 2D4) generated using immunogen VDP-1 (SEQ ID NO: 14) solve this long-standing problem.

EXAMPLE 4

(The sequences and CDR’s of the 2D4 antibody were characterized”

The specificity of an antibody is primarily defined by the amino acid sequence of the complementarity-determining regions (CDRs) present within the hyper-variable region of the heavy chains and light chains that comprise an antibody. Residues within the CDRs make direct contact with the antigen and the CDRs have shape complementation with the antigen, and can thus be used to facilitate engineering of synthetic antibodies with the same specificity, as is known to those skilled in the art.

In this example, antibody sequences were determined as described in Example 1 above, and are summarized in Table 4 below.

According to aspects of the present invention, the heavy chain 2D4 sequence (SEQ ID NO:19) comprises the heavy chain CDRH1 , CDRH2 and CDRH3 sequences, and the light chain 2D4 sequence (SEQ ID NO: 17) comprises the light chain CDRL1 , CDRL2 and CDRL3 sequences.

According to additional aspects of the present invention, the variable heavy and variable light chains provide means for positioning their respective CDRs in a configuration that binds to variably deamidated gluten from wheat, barley or rye.

According to further aspects of the present invention, SEQ ID NO: 19 and SEQ ID NO: 17 each define one representative means for positioning the heavy chain CDRHs and the light chain CDRLs, respectively, in a configuration that binds to variably deamidated gluten from wheat, barley or rye.

Table 4: 2D4 Variable region aa sequences.

Tables 5 and 6 summarize CDR1 , CDR2 and CDR3 regions for both the heavy and light variable regions, respectively, derived by use of various art-recognized numbering/modeling tools summarized in Table 7. Collectively, these CDRs define the specificity of an antibody (e.g., 2D4), and confer its unique ability to recognize gluten epitopes that are native or variably deamidated. Table 5: Heavy chain CDR1 , CDR2 and CDR3 aa sequences.

Table 6: Light chain CDR1 , CDR2 and CDR3 aa sequences.

Table 7: Numbering and CDR assignment/modeling tools.

EXAMPLE 5

(The exemplary the 2D4 antibody was used in exemplary Western blot methods and kits)

The exemplary mAb 2D4 was assessed in Western blot using native prolamins derived from wheat, barley, rye, and oats as depicted in Figures 3A, 3B and 3C. The art-recognized mAb R5 was used as a comparator.

Specifically, Figures 3A-3C show SDS-PAGE and corresponding Western blot analysis of native prolamins. Figure 3A shows SDS-PAGE analysis of wheat gliadin (“Gli”), barley hordein (“Hor”), rye secalin (“Sec”), and oat avenin (“Ave”), electrophoretically separated under reducing (denaturing) conditions. Protein bands for each prolamin are also displayed. Figure 3B and 3C show comparative Western blot analysis R5 (art-recognized standard) and 2D4, respectively, blotted against prolamins: wheat gliadin (“Gli”), barley hordein (“Hor”), rye secalin (“Sec”), and R5(-) oat avenin (“Ave”), electrophoretically separated under reducing (denaturing) conditions. “MK” refers to the molecular weight marker values in kDa units. Both antibodies detected the a-p-y-fractions (ranging from 32 to 45 kDa), and co-gliadin fractions (ranging from 43-68 kDa) from wheat, as well as the B-(ranging from 30-50 kDa), C-(ranging from 55 to 86 kDa), and y-hordeins (ranging from 25-55 kDa) from barley, and the y75 (ranging from 54 to 66 kDa), y40 (ranging from 33 to 37 kDa), and co-secalins (ranging from 45-50 kDa) from rye. Similarly, both monoclonal antibodies exhibited limited reactivity with D- hordeins (ranging from 85-97 kDa). Neither antibody exhibited binding with avenins from oats. The banding profile of 2D4 was, therefore, qualitatively similar to that of R5, indicating similar reactivity towards the different native gluten species and suitability of 2D4 for use in detecting native gluten using Western blot analysis.

Western blot analysis was also performed using deamidated prolamins from wheat to further assess exemplary mAb 2D4. Specifically, Figures 4A-4C show SDS- PAGE and corresponding Western blot analysis of deamidated gliadins from wheat electrophoretically separated under reducing (denaturing) SDS-PAGE conditions (Fig. 4A). Figures 4B and 4C show comparative Western blot analysis comparison of R5 IgG and 2D4 IgG, respectively, blotted against: lane 1 ) Native gliadin, lane 2) 25-30% deamidated acid hydrolyzed wheat protein sample 1 , lane 3) 50% deamidated hydrolyzed wheat protein, lane 4) 50-60% deamidated acid hydrolyzed wheat protein GluPearl-19S, lane 5) 50-60% deamidated hydrolyzed wheat protein sample 3, lane 6) 60% deamidated acid hydrolyzed wheat protein sample 2, lane 7) 1 h Protein glutaminase-treated sample (20% deamidated), lane 8) 5 h Protein glutaminase-treated sample (60% deamidated), and lane 9) 30 h Protein glutaminase-treated sample (72% deamidated). “Mk” refers to molecular weight marker values in kDa units. For all deamidated gliadin samples, there was limited definition of bands compared with the native gluten (NG) sample. The more severely deamidated samples (>50% deamidated) produced under acid treatment demonstrated highly atypical (relative to native) appearance on SDS-PAGE, presumably due to pronounced proteolysis (Figure 4A). R5 immunoblotting (Figure 4B) was very faintly positive for three of the eight samples including 25-30% deamidated acid hydrolyzed wheat protein sample 1 (lane 2) and 50% deamidated (lane 3), and the 20% deamidated 1 h treatment of gluten with protein glutaminase (lane 7). By contrast, mAb 2D4 immunoblotting (Figure 4C) resulted in much more pronounced binding, with positive outcomes for five of the eight samples 25-30% deamidated acid hydrolyzed wheat protein sample 1 (lane 2), 50% deamidated (lane 3), 50-60% deamidated acid hydrolyzed wheat protein GluPearl-19S (lane 4), 60% deamidated acid hydrolyzed wheat protein sample 2 (lane 6), and the 20% deamidated 1 h treatment of gluten with protein glutaminase (lane 7). The mAb 2D4, therefore, exhibited superior ability to bind increasingly deamidated wheat protein samples compared with mAb R5. Differences in staining patterns between the SDS- PAGE and the Western blot were noted. Comparison of 2D4 and R5 antibodies in Western blot revealed qualitatively similar profiles for native gliadin (lane 1 ). In the deamidated samples, most of the lower molecular weight protein (<40KDa) was not detected by either antibody, presumably due to the high degree of hydrolysis. However, mAb 2D4 was able to detect some higher molecular weight proteins which were undetected by R5. In summary, 2D4 has significantly increased reactivity with deamidated gluten compared with R5, thereby indicating suitability of exemplary mAb 2D4 for use in detecting deamidated gluten using Western blot analysis.

EXAMPLE 6

(The exemplary the 2D4 antibody was used in indirect ELISA methods and kits)

The exemplary mAb 2D4 was assessed in indirect ELISA methods. Exemplary mAb 2D4 was assessed for its ability to detect gluten in indirect ELISA, an embodiment of the kit of the invention. As shown above in Figure 1 , mAb 2D4 exhibited improved affinity towards deamidated gliadins compared to mAb R5, with no significant difference in its ability to detect native gluten. The mAb 2D4 was also assessed using a panel of plate-bound native gliadin, hordein, secalin, and avenins (prolamins from oats), as well as protein extracts derived from soy, millet, com, and rice, using the mAb R5 as a comparator (Figure 5). Specifically, Figures 5A and 5B show indirect ELISA-based analysis of the antibodies against unmodified prolamins and other proteins. The target proteins (5 pg/mL) (soy protein, as well as a panel of prolamins including wheat gliadin, barley hordein, rye secalin, and R5(-) oat avenins, as well as protein extracts from rice, corn, soy, and millet) were immobilized on the well surface. Figures 5A and 5B show comparative results with antibodies R5 (art-recognized standard) and 2D4, respectively, diluted and titrated to assess their relative binding profile against the target proteins. OD values at 450 nm as reported as a function of antibody concentration are graphically represented.

Significantly, in this ELISA format, mAb 2D4 exhibited near-equivalent reactivity against native gliadins, hordeins, and secalins, with half-binding concentrations graphically estimated at 0.029, 0.034, and 0.045 pg/mL for gliadins, hordeins, and secalins respectively. Additionally, mAb 2D4 exhibited very weak reactivity against avenins, and no reactivity against corn, rice, or soy extracts. The comparator, mAb R5, demonstrated half-binding concentrations that were essentially identical to those of mAb 2D4. Collectively, these results, along with the results of Examples 2 and 5, establish the suitability of exemplary mAb 2D4 for use in detecting native and deamidated gluten, including in indirect ELISA methods and kits.

EXAMPLE 7

(The exemplary the 2D4 antibody was used in sandwich ELISA methods and kits)

Exemplary mAb 2D4, was assessed for its ability to detect native and deamidated gluten in Sandwich ELISA format, an exemplary kit embodiment of the invention. The sandwich format is a preferred ELISA format used by most test systems in the food diagnostic industry. The kits used in this analysis included: a 2D4-based kit (based on the 2D4 antibody), R-Biopharm RIDASCREEN™ Gliadin (based on the R5 antibody), and Romer AgraQuant™ Gluten G12 (based on the G12 antibody). Native gliadin (chemically unmodified) was used as a control and was similarly detected by all three kits. Although the deamidation process may be accompanied by significant proteolysis (e.g., as depicted in Figure 4), which hinders detection using the sandwich ELISA format, the 2D4-based kit consistently out-performed the R5 and G12 kits in its ability to detect the deamidated samples, in a manner that was consistent with the degree of sample deamidation (Table 2). In summary, mAb 2D4 was shown to be suitable for use in sandwich ELISA kits for detecting/quantifying native and deamidated gluten.

Table 2. Comparative gliadin concentrations obtained using Sandwich ELISA*

EXAMPLE 8

(The exemplary the 2D4 antibody was used in Immunochromatographic/lateral flow methods and kits)

The exemplary mAb 2D4 was assessed for its ability to detect native gluten in a lateral flow assay (immunochromatographic technique), another exemplary kit embodiment of the invention.

A schematic of the assay and its interpretation are depicted in Figures 6A and 6B. In the assay, the mAb 2D4 is configured in sandwich format and in competitive format, thereby enabling the semi-quantitative determination of gluten present in a food, personal care product, or on environmental surfaces.

Specifically, Figures 6A and 6B show a schematic for a lateral flow device used to detect gluten. As shown in Fig. 6A, the test features an antibody directed against variably deamidated gluten, wherein the antibody (2D4 in this example) is configured in sandwich format and competitive format zones. Gluten present in the test sample/solution (introduced at the sample port) reacts with 2D4-conjugated gold colloid present within the test device to form a conjugate-gluten complex. The complex then serially wicks across the reagent zones by capillary action. The reagent zones include a sandwich test line (“T”) consisting of immobilized 2D4, a competitive test line (“0”) consisting of immobilized antigen, and a control line (C) consisting of a secondary antibody with specificity for mouse IgG. Upon entering the reagent zones, conjugate- gluten complex, if present, will attach to the sandwich test line, causing the appearance of a red (shown here in gray scale) sandwich test line in the reagent zone. The complex not sequestered at the sandwich test line, will additionally compete for immobilized antigen at the competitive test line, resulting in the disappearance of the red (shown here in gray scale) competitive test line. The procedural control is used to confirm assay functionality, causing the appearance of a red (shown here in gray scale) control line in the reagent zone. The outcome is visually interpreted at 10-15 minutes. As illustrated in Fig. 6B, the interpretation of the test is as follows: the sandwich test line (“T”) will become clearly visible at the limit of detection value or the target analyte concentration up to a point where it will then start to fade and eventually disappear at high analyte levels. The competitive test line (“0”) will be intense if the sample is negative for the target gluten analyte and fade with increasing amounts of target gluten analyte, disappearing completely at high analyte levels before the Sandwich Test Line does. This feature allows the operator to distinguish between samples with none or low levels of target analyte and those with high levels. The competitive test line also enables detection of target analyte that is highly hydrolyzed and therefore poorly detected by the sandwich test line. Failure of the procedural control line (“C”) to appear denotes an invalid test, requiring repeat testing. Table 3 summarizes testing outcomes using a lateral flow test based in the 2D4 antibody to measure gluten spiked into a rice cereal matrix at different contamination concentrations (0, 3, 8, 15, 25 mg/kg gluten) in terms of probability of detection (POD), and compares these outcomes against values obtained using the AOAC OMA reference method (based on the R5 antibody, supplied as an ELISA kit) on the same samples. This example establishes the suitability of 2D4 for use in LFD kits for detecting/quantifying native gluten

Table 3. Gluten detection using a lateral flow device (LFD) configured with antibody 2D4, compared against that of AOAC Reference Method OMA 2012.01 , an ELISA kit based on the art-recognized antibody R5.

AOAC OMA

^e

2012.01

a N = Number of test portions b X = Number of positive test portions c POD = Positive outcomes divided by the total number of trials d 95% Cl = 95% Confidence Intervals e OMA = Official Method of Analysis

EXAMPLE 9

(The exemplary 2D4 antibody was used in Immunochromatographic/lateral flow methods and kits to detect deamidated gluten in food and cosmetic products)

The exemplary mAb 2D4 was assessed for its ability to detect deamidated gluten in a lateral flow assay (immunochromatographic technique), another exemplary kit embodiment of the invention.

Table 8 summarizes testing outcomes using a lateral flow test based in the 2D4 antibody to measure deamidated gluten naturally found in food and cosmetic products in the form of hydrolyzed wheat protein (HWP) or enzymatically processed (malted barley vinegar, comparing these outcomes against values obtained using the AOAC OMA reference method (ELISA, based on the R5 antibody) on the same samples. This example establishes the suitability of 2D4 for use in LFD kits for detecting/quantifying deamidated gluten in food and cosmetic products.

Applicant notes that according to FDA, Health Canada, the European Union, and the Food and Agriculture Organization of the United Nations (FAO), foods exceeding a threshold of 20 mg/kg (ppm) do not meet the regulatory standards to qualify as gluten- free and are subject to penalty if indicated as gluten-free on the packaging. Likewise, according to Gluten-Free Certification Organization (GFCO), any food exceeding a threshold of 10 mg/kg (ppm) does not qualify for gluten-free certification and therefore cannot display trademark Gluten Free labeling on the packaging. Table 8. Deamidated gluten detection in food and cosmetic products using a lateral flow device (LFD) configured with antibody 2D4, compared against that of AOAC Reference Method OMA 2012.01 based on the art-recognized antibody R5. 2D4 LFD AOAC OMIVIAM^a

2012.01

a OMA = Official Method of Analysis (ELISA) based on R5

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Claims

1 . An isolated antigen binding protein that binds to variably deamidated gluten from wheat, barley or rye, comprising: a) a heavy chain polypeptide having the following complementary determining regions (CDRs): a CDR1 from a CDRH1 in SEQ ID NO:19, a CDR2 from a CDRH2 in SEQ ID NO:19, and a CDR3 from a CDRH3 in SEQ ID NO:19; and b) a light chain polypeptide having the following CDRs: a CDR1 from a CDRL1 in SEQ ID NO:17, a CDR2 from a CDRL2 in SEQ ID NO:17, and a CDR3 from a CDRL3 in SEQ ID NO:17.

2. The antigen binding protein of claim 1 , wherein: the heavy chain CDR1 in SEQ ID NO: 19 is selected from: SEQ ID NOS:27, 30,

32, 38, or 42, the heavy chain CDR2 in SEQ ID NO: 19 is selected from: SEQ ID NOS:28, 31 ,

33, 37, 39, 41 , 43, or 45, and the heavy chain CDR3 in SEQ ID NO: 19 is selected from: SEQ ID NOS:29, 40, or 44; and the light chain CDR1 in SEQ ID NO:17 is selected from: SEQ ID NOS:21 , 24, or

34, the light chain CDR2 in SEQ ID NO:17 is selected from: SEQ ID NOS:22, 25, 26, or 35, and the light chain CDR3 in SEQ ID NO:17 is selected from: SEQ ID NOS:23, or 36.

3. The antigen binding protein of 2, comprising one of the following sets of complementary-determining regions (CDRs):

(i) heavy chain CDR1 , CDR2, CDR3 and light chain CDR1 , CDR2 and CDR3 having the amino acid sequences AASGFTLSTYAMS (SEQ ID NO:27), SISRSGDTYKGR (SEQ ID NO:28), ATLYYYGSTWYFDV (SEQ ID NO:29),and SASQDINNYLN (SEQ ID NO:21 ), YYTSSLHS (SEQ ID NO:22), QQYSKLPWT, (SEQ ID NO:23), respectively; or (ii) heavy chain CDR1 , CDR2, CDR3 and light chain CDR1 , CDR2 and CDR3 having the amino acid sequences GFTLSTYA (SEQ ID NO:30), ISRSGDTYKG (SEQ ID NO:31 ), ATLYYYGSTWYFDV (SEQ ID NO:29), and QDINNY (SEQ ID NO:24), YTS (SEQ ID NO:25), QQYSKLPWT (SEQ ID NO:23), respectively; or

(iii) heavy chain CDR1 , CDR2, CDR3 and light chain CDR1 , CDR2 and CDR3 having the amino acid sequences GFTLSTY (SEQ ID NO:32), SRSGDTYK (SEQ ID NO:33), ATLYYYGSTWYFDV (SEQ ID NO:29), and SASQDINNYLN (SEQ ID NO:21 ), YTSSLHS (SEQ ID NO:26), QQYSKLPWT (SEQ ID NO:23), respectively; or

(iv) heavy chain CDR1 , CDR2, CDR3 and light chain CDR1 , CDR2 and CDR3 having the amino acid sequences GFTLSTYA (SEQ ID NQ:30), ISRSGDT (SEQ ID NO:37), ATLYYYGSTWYFDV (SEQ ID NO:29), and QDINNY (SEQ ID NO:24), YTS (SEQ ID NO:25), QQYSKLPWT (SEQ ID NO:23), respectively; or

(v) heavy chain CDR1 , CDR2, CDR3 and light chain CDR1 , CDR2 and CDR3 having the amino acid sequences TYAMS (SEQ ID NO:38), SISRSGDTYKGRYPD (SEQ ID NO:39), LYYYGSTWYFDV (SEQ ID NQ:40),and SASQDINNYLN (SEQ ID NO:21 ), YTSSLHS (SEQ ID NO:26), QQYSKLPWT (SEQ ID NO:23), respectively; or

(vi) heavy chain CDR1 , CDR2, CDR3 and light chain CDR1 , CDR2 and CDR3 having the amino acid sequences GFTLSTY (SEQ ID NO:32), SRSGD (SEQ ID NO:41 ), LYYYGSTWYFDV (SEQ ID NQ:40), and SASQDINNYLN (SEQ ID NO:21 ), YTSSLHS (SEQ ID NO:26), QQYSKLPWT (SEQ ID NO:23), respectively; or

(vii) heavy chain CDR1 , CDR2, CDR3 and light chain CDR1 , CDR2 and CDR3 having the amino acid sequences STYAMS (SEQ ID NO:42), WVTSISRSGDTY (SEQ ID NO:43), ATLYYYGSTWYFD (SEQ ID NO:44), and NNYLNWY (SEQ ID NO:34), LLIYYTSSLH (SEQ ID NO:35), QQYSKLPW (SEQ ID NO:36), respectively; or

(viii) heavy chain CDR1 , CDR2, CDR3 and light chain CDR1 , CDR2 and CDR3 having the amino acid sequences TYAMS (SEQ ID NO:38), SISRSGDTYKGRYPDS (SEQ ID NO:45), LYYYGSTWYFDV (SEQ ID NQ:40), and SASQDINNYLN (SEQ ID NO:21 ), YTSSLHS (SEQ ID NO:26), QQYSKLPWT (SEQ ID NO:23), respectively.

4. The antigen-binding molecule of any one of claims 1-3, selected from a monoclonal antibody and/or an antigen-binding fragment thereof.

5. The antigen-binding molecule of claim 4, wherein the antigen-binding molecule is a murine antibody of isotype lgG1 kappa.

6. An isolated antigen binding protein, comprising: a) a heavy chain CDR1 selected from SEQ ID NOS:27, 30, 32, 38, or 42, a heavy chain CDR2 selected from SEQ ID NOS:28, 31 , 33, 37, 39, 41 , 43, or 45, and a heavy chain CDR3 selected from SEQ ID NOS:29, 40, or 44; b) a light chain CDR1 selected from SEQ ID NOS:21 , 24, or 34, a light chain CDR2 selected from SEQ ID NOS:22, 25, 26, or 35, and a light chain CDR3 selected from SEQ ID NOS:23, or 36; and c) means for positioning the CDRs of a) and b) in a configuration that binds to variably deamidated gluten from wheat, barley or rye.

7. The isolated antigen binding protein of claim 6, comprising one of the following sets of complementary-determining regions (CDRs):

(i) heavy chain CDR1 , CDR2, CDR3 and light chain CDR1 , CDR2 and CDR3 having the amino acid sequences AASGFTLSTYAMS (SEQ ID NO:27), SISRSGDTYKGR (SEQ ID NO:28), ATLYYYGSTWYFDV (SEQ ID NO:29),and SASQDINNYLN (SEQ ID NO:21 ), YYTSSLHS (SEQ ID NO:22), QQYSKLPWT, (SEQ ID NO:23), respectively; or

(ii) heavy chain CDR1 , CDR2, CDR3 and light chain CDR1 , CDR2 and CDR3 having the amino acid sequences GFTLSTYA (SEQ ID NQ:30), ISRSGDTYKG (SEQ ID NO:31 ), ATLYYYGSTWYFDV (SEQ ID NO:29), and QDINNY (SEQ ID NO:24), YTS (SEQ ID NO:25), QQYSKLPWT (SEQ ID NO:23), respectively; or

(iii) heavy chain CDR1 , CDR2, CDR3 and light chain CDR1 , CDR2 and CDR3 having the amino acid sequences GFTLSTY (SEQ ID NO:32), SRSGDTYK (SEQ ID NO:33), ATLYYYGSTWYFDV (SEQ ID NO:29), and SASQDINNYLN (SEQ ID NO:21 ), YTSSLHS (SEQ ID NO:26), QQYSKLPWT (SEQ ID NO:23), respectively; or

(iv) heavy chain CDR1 , CDR2, CDR3 and light chain CDR1 , CDR2 and CDR3 having the amino acid sequences GFTLSTYA (SEQ ID NQ:30), ISRSGDT (SEQ ID NO:37), ATLYYYGSTWYFDV (SEQ ID NO:29), and QDINNY (SEQ ID NO:24), YTS (SEQ ID NO:25), QQYSKLPWT (SEQ ID NO:23), respectively; or

(v) heavy chain CDR1 , CDR2, CDR3 and light chain CDR1 , CDR2 and CDR3 having the amino acid sequences TYAMS (SEQ ID NO:38), SISRSGDTYKGRYPD (SEQ ID NO:39), LYYYGSTWYFDV (SEQ ID NQ:40),and SASQDINNYLN (SEQ ID N0:21 ), YTSSLHS (SEQ ID NO:26), QQYSKLPWT (SEQ ID NO:23), respectively; or

(vi) heavy chain CDR1 , CDR2, CDR3 and light chain CDR1 , CDR2 and CDR3 having the amino acid sequences GFTLSTY (SEQ ID NO:32), SRSGD (SEQ ID NO:41), LYYYGSTWYFDV (SEQ ID NQ:40), and SASQDINNYLN (SEQ ID NO:21 ), YTSSLHS (SEQ ID NO:26), QQYSKLPWT (SEQ ID NO:23), respectively; or

(vii) heavy chain CDR1 , CDR2, CDR3 and light chain CDR1 , CDR2 and CDR3 having the amino acid sequences STYAMS (SEQ ID NO:42), WVTSISRSGDTY (SEQ ID NO:43), ATLYYYGSTWYFD (SEQ ID NO:44), and NNYLNWY (SEQ ID NO:34), LLIYYTSSLH (SEQ ID NO:35), QQYSKLPW (SEQ ID NO:36), respectively; or

(viii) heavy chain CDR1 , CDR2, CDR3 and light chain CDR1 , CDR2 and CDR3 having the amino acid sequences TYAMS (SEQ ID NO:38), SISRSGDTYKGRYPDS (SEQ ID NO:45), LYYYGSTWYFDV (SEQ ID NQ:40), and SASQDINNYLN (SEQ ID NO:21 ), YTSSLHS (SEQ ID NO:26), QQYSKLPWT (SEQ ID NO:23), respectively.

8. The antigen-binding molecule of claim 6, wherein the means for positioning comprises a light chain polypeptide comprising the light chain CDRs and a heavy chain polypeptide comprising the heavy chain CDRs, preferably wherein the means for positioning comprises: a heavy chain polypeptide SEQ ID NO: 19 having the heavy chain CDRs; and a light chain polypeptide SEQ ID NO: 17 having the light chain CDRs.

9. The antigen-binding molecule of claim 7, wherein the means for positioning comprises a light chain polypeptide comprising the light chain CDRs and a heavy chain polypeptide comprising the heavy chain CDRs, preferably wherein the means for positioning comprises: a heavy chain polypeptide SEQ ID NO: 19 having the heavy chain CDRs; and a light chain polypeptide SEQ ID NO: 17 having the light chain CDRs.

10. The antigen-binding molecule of any one of claims 6-9, selected from a monoclonal antibody and/or an antigen binding fragment thereof.

11 . The antigen-binding molecule of claim 10, wherein the antigen-binding molecule is a murine antibody of isotype lgG1 kappa.

12. A cell expressing the antigen binding protein of any one of claims 1-11.

13. The cell of claim 12, wherein the cell is a hybridoma, or other suitable immortalized cell line.

14. A kit, combination, or composition comprising the antigen binding protein of any one of claims 1-11.

15. A method for detecting, enriching, and/or quantifying variably deamidated gluten, comprising: contacting a sample, or component thereof, suspected of containing variably deamidated gluten from wheat, barley and/or rye with the antigen binding protein of any one of claims 1-11 ; and detecting, enriching, and/or quantifying the variably deamidated gluten based on the contacting.

16. The method of claim 15, wherein the contacting comprises forming a complex with the antigen binding protein and the gluten, and detecting, enriching, and/or quantifying the variably deamidated gluten based directly or indirectly on the complex.

17. The method of claim 16, wherein the method is selected from the group consisting of Western blot analysis, indirect ELISA, sandwich ELISA, lateral flow, and combinations thereof.

18. The method of any one of claims 15-17, wherein the sample, or component thereof, is selected from food, personal care products, or human biological samples.

19. An isolated polypeptide selected from the group consisting of VDP-2, VDP-3 and VDP-4 having the sequences LZPZQPFPZZLZPZZPFPZZ (SEQ ID NO:46), LZPZZPFPZZLZPZQPFPZZ (SEQ ID NO:47), LZPZQPFPZZLZPZQPFPZZ (SEQ ID NO:48), respectively, concatenates and/or mixed concatenates thereof, and combinations thereof.

20. The isolated polypeptide of claim 19, wherein the polypeptide is substituted at an N-terminal and/or C-terminal end.

21 . The isolated polypeptide of claim 20, wherein the N-terminal and/or C- terminal substituent is one or more label selected from the group consisting of biotin, an enzyme, a fluorogenic substance, a radioactive substance, a chemiluminescent substance, carrier protein, membrane-active peptide (MAP), an electrogenerated chemiluminescent substance, a nanoparticle, PEGylation, and combinations thereof.

22. A kit, combination, or composition comprising the isolated polypeptide of any one of claims 19-21.

23. The kit or combination of claim 22, further comprising an antibody that binds to the polypeptide.

24. A method of generating and selecting an antibody producing cell, comprising: immunizing an animal with a synthetic variably deamidated polypeptide selected from the group consisting of VDP-1 , VDP-2, VDP-3 and VDP-4 having the sequences LZPZZPFPZZLZPZZPFPZZ (SEQ ID NO: 14), LZPZQPFPZZLZPZZPFPZZ (SEQ ID NO:46), LZPZZPFPZZLZPZQPFPZZ (SEQ ID NO:47), LZPZQPFPZZLZPZQPFPZZ (SEQ ID NO:48), respectively, concatenates and/or mixed concatenates thereof, and combinations thereof; and generating an antibody producing cell from lymphocytes of the immunized animal, wherein the produced antibody binds to both native gluten and variably deamidated gluten from wheat, barley, and rye.

25. The method of claim 24, wherein synthetic variably deamidated polypeptide is selected from the group consisting of VDP-2, VDP-3 and VDP-4 having the sequences LZPZQPFPZZLZPZZPFPZZ (SEQ ID NO:46), LZPZZPFPZZLZPZQPFPZZ (SEQ ID NO:47), LZPZQPFPZZLZPZQPFPZZ (SEQ ID NO:48), respectively, concatenates and/or mixed concatenates thereof, and combinations thereof.

26. The method of claim 24 or 25, wherein the immunization comprises boosting with the synthetic variably deamidated polypeptide and/or with variably deamidated gluten from wheat, barley, and/or rye.

27. The method of claim 26, wherein the animal is: initially immunized with the synthetic variably deamidated polypeptide selected from the group consisting of VDP-1 , VDP-2, VDP-3 and VDP-4 having the sequences LZPZZPFPZZLZPZZPFPZZ (SEQ ID NO: 14), LZPZQPFPZZLZPZZPFPZZ (SEQ ID NO:46), LZPZZPFPZZLZPZQPFPZZ (SEQ ID NO:47), LZPZQPFPZZLZPZQPFPZZ (SEQ ID NO:48), respectively, concatenates and/or mixed concatenates thereof, and combinations thereof; and subsequently boosted with the synthetic variably deamidated polypeptide and/or with variably deamidated gluten from wheat, barley, and/or rye.

28. The method of claim 26, wherein the animal is: initially immunized with the synthetic variably deamidated polypeptide selected from the group consisting VDP-2, VDP-3 and VDP-4 having the sequences

LZPZQPFPZZLZPZZPFPZZ (SEQ ID NO:46), LZPZZPFPZZLZPZQPFPZZ (SEQ ID NO:47), LZPZQPFPZZLZPZQPFPZZ (SEQ ID NO:48), respectively, concatenates and/or mixed concatenates thereof, and combinations thereof; and subsequently boosted with the synthetic variably deamidated polypeptide and/or with variably deamidated gluten from wheat, barley, and/or rye.

29. The method of any one of claims 24-28, wherein the animal is a mouse or a rat.

30. The method of any one of claims 24-29, wherein the antibody producing cell is a hybridoma, or other suitable immortalized cell.