NZ614464B2 - Methods and apparatus for detection of gluten sensitivity, and its differentiation from celiac disease - Google Patents

Abstract

Disclosed is a method of characterising the antibody composition of a sample from an animal, comprising: measuring a first signal derived from binding of a first antibody of the sample to whole-wheat antigen obtainable by combining water-soluble proteins and alcohol-soluble proteins extracted from wheat; measuring a second signal derived from binding of a second antibody of the sample to a gliadin antigen; and measuring a third signal derived from binding of a third antibody of the sample to an antigen selected from the group consisting of: (1) a wheat germ agglutinin; (2) gluteomorphin; (3) a glutenin; (4) a deamidated glutenin; (5) a prodynorphin; and (6) a dynorphin, wherein the presence of the first, second, and third signals indicate the presence of the first, second and third antibodies in the sample, respectively. m wheat; measuring a second signal derived from binding of a second antibody of the sample to a gliadin antigen; and measuring a third signal derived from binding of a third antibody of the sample to an antigen selected from the group consisting of: (1) a wheat germ agglutinin; (2) gluteomorphin; (3) a glutenin; (4) a deamidated glutenin; (5) a prodynorphin; and (6) a dynorphin, wherein the presence of the first, second, and third signals indicate the presence of the first, second and third antibodies in the sample, respectively.

Description

/021892 METHODS AND APPARATUS FOR DETECTION OF GLUTEN SENSITIVITY, AND ITS DIFFERENTIATION FROM CELIAC DISEASE RELATED APPLICATION The present application claims the benefit of US. Provisional Application No. 61/434,501 filed January 20, 2011, which is incorporated herein in its entirety by reference.

FIELD OF THE INVENTION The present invention relates to methods and kits for aid in diagnosis of gut-related IO es and pathologies, including at least gluten immune reactivity and sensitivity, silent celiac disease, and Crohn’s disease.

BACKGROUND OF THE INVENTION Wheat allergy, celiac disease and gluten sensitivity are three distinct conditions that are triggered by the ingestion of wheat gliadin (1, 2). In these conditions, the on to gluten is mediated by both cellular and humoral immune responses, ing in the tation of ent matologies. For example, in wheat y a specific cells sequence of gliadin peptides cross-links two IgE molecules on the surface of mast and basophils that trigger the release of mediators such as histarnines and leukotrienes (3).

These and all other extrinsic materials discussed herein are incorporated by reference in their entirety. Where a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.

Celiac disease (CD) is an autoimmune condition with known genetic makeup and environmental triggers, such as gliadin peptides. CD affects between 1-2% of the general population. hout this application, unless the context dictates the contrary, all ranges set forth herein should be reted as being inclusive of their endpoints, and open~ended all lists ranges should be interpreted to include commercially practical values. Similarly, of values should be considered as inclusive of intermediate values unless the context indicates the ry.

Markers for confirming a sis of this disorder are IgA against native, deamidated gliadin peptides and IgA anti-tissue transglutaminase (tTg) autoantibcdy. In PCT/U52012/021892 comparison with CD, gluten sensitivity (GS) affects up to 30% of the population (4).

According to two articles published in 2010 and 2011 by Sapone et al (5, 6), symptoms in GS may resemble some of the gastrointestinal symptoms that are associated with CD or wheat allergy, but it is emphasized that ive diagnostic tests for gluten sensitivity are currently missing (5, 6). While studying the innate and immune responses in CD compared to those in GS, the researchers found that TLRl, TLR2 and TLR4, which are associated with innate immunity, were elevated in mucosal GS but not in CD, while biomarkers of adaptive immunity such as lFN-g, lL—21 and IL-17A were sed in mucosal tissue in CD but not GS. They believed that measurements of toll-like receptors and IFN-y, lL-21 and IL-17A would enable them to differentiate between CD and GS (5, 6) with a method that is highly invasive and would require a biopsy. Immediate type hypersensitivity to gluten is IgE mediated, while delayed type ensitivity to gluten is an antibody- (IgG, IgA) and T-cell-mediated reaction, which is called celiac disease or gluten sensitivity with pathy (7). In the absence of IgG and IgA against tTg, elevated IgG and IgA against various wheat antigens and peptides indicate the loss of mucosal immune tolerance against wheat peptides and the pment of gluten sensitivity (7). Due to antigenic similarities n wheat antigens and human tissue, both CD and GS can result in many autoimmune conditions, including Type 1 diabetes, arthritis, thyroiditis, and even neuroautoimmune conditions such gluten ataxia and multiple sclerosis .

It should be appreciated that the term “patients” refer to humans under the care of a health care professional. More broadly, r, the novel testing protocols and analyses disclosed herein could be applied to nonspatient humans, and any other animal that could suffer from celiac disease, gluten sensitivity, and gut-related autoimmunities.

While GS patients, similar to CD patients, are unable to te gluten and can develop the same or similar sets of gastrointestinal symptoms, in GS this immune reaction does not lead to small intestine damage (5, 6). This lack of induction of intestinal damage in GS and the association of CD with genetic markers HLA DQ2/DQ8 plus small intestinal damage make the diagnosis of CD much easier than GS. The less severe clinical e in GS, the absence of tTg autoantibodies, and the dismissal of the significance of elevated IgG and IgA autoantibodies against various wheat proteins and peptides by many ians makes GS an extremely ous disorder. This is because the persistence of IgG and/or IgA antibodies in the blood for long s of time, along with inducers of inflammatory cascades can result in full-blown autoimmunity. If this were to be the case, WO 00070 PCT/U82012/021892 due to the severity of the resulting tissue damage even implementation of a gluten-free diet might not be able to help reverse the course of the autoimmune reaction induced by IgG and IgA antibodies against different wheat antigens and peptides.

A comparison between celiac disease and gluten immune reactivity/sensitivity is shown in Figure 1. According to this model, if two children, one with a negative genetic makeup (HLA DQ2/DQ8'), and the other with positive (HLA DQ2/DQ8+), are exposed to environmental factors, such as Rota virus, bacterial endotoxins, and some medications or their synergistic effects, the result can be a breakdown of mucosal immune tolerance in both children. The induction of mucosal immune tolerance against n results in the IO production of IgA and/or IgG against native wheat proteins and peptides, which is the next step in the initiation of gluten sensitivity in both individuals that are HLA DQ2/DQ8‘ HLA DQ2/DQ3".

However, in the individual with the positive genetic makeup, the IgG and IgA antibodies against gliadin along with biomarkers of inflammation can activate tTg, induce damage to the villi, and result in villous y. ation of a specific gliadin peptide leads to the formation of a complex between it and the tTg; the tation of this complex by n-presenting cells to T cells and B cells results in IgA or IgG production t tTg, deamidated gliadin and the gliadin-tTg complex. The formation of these antibodies and their detection in blood is the hallmark of CD, which is an inherited condition detected in 1-2% of the population. If CD is left untreated, the outcome could be autoimmunities and .

In comparison, in an individual negative for HLA DQ2/DQ8, this breakdown in immunological tolerance and the concomitant production of IgA and or IgG against native wheat ns and peptides may activate an inflammatory cascade. In the absence of tTg activation, however, s atrophy does not occur. Furthermore, gliadin peptides do not and IgA antibodies are produced only go through de—amidation, and consequently IgG against native wheat and gliadin peptides.

With continuous re to wheat antigens and uous l immune tolerance, the wheat antigens and reacting antibodies form an unholy alliance of immune complexes, resulting in severe gluten immune reactivity and ivity. This immune reactivity and sensitivity is a non-inherited condition detected in up to 30% of the population. If this disorder is left unchecked, prolonged exposure to IgG and igA antibodies against wheat antigens and peptides and their cross-reaction with different tissue antigens can result in various autoimmune disorders. Therefore, even in the absence of CD, GS might still provide a productive environment for other gluten-related autoantibodies that attack different organs.

Furthermore, a gluten-free diet usually is only recommended for those who meet the criteria for a sis of CD, not of gluten immune reactivity and sensitivity. unately, that leaves many gluten-sensitive people ing unnecessarily with very s symptoms that put them at risk for complications, conditions that might be resolved with a -free diet, if they only knew.

Thus, a new paradigm is needed for aid in diagnosing and distinguishing among various lated diseases, including gluten immune reactivity and sensitivitypsilent celiac disease, celiac disease, and gut—related autoimmunity.

OBJECT It is an object of the present invention to provide a method of characterizing the antibody composition of a sample from an animal, a method of generating an antibody profile, a method of screening a sample from an animal, a method of screening for the presence of IgG and/or IgA antibodies to Wheat antigens in a sample from an animal, a test plate and/or a test kit that overcome or ameliorate at least one of the disadvantages of the prior art. Any objects referred to herein should be read disjunctively and with the alternative object of to at least provide the public with a useful choice.

SUMMARY OF THE INVENTION The inventive subject matter of the present invention provides apparatus, systems and methods in which antibodies are used as biomarkers to assist in diagnosing gluten immune reactivity and ivity, silent celiac disease, Crohn's disease and other gut—related pathologies.

In n aspects of the present ion, whole blood, blood sera, saliva or other samples from a human or other animal are tested for dies to (a) a wheat antigen; (b) a gliadin antigen; and (0) one or more of a wheat germ agglutinin, a gluteomorphin, a glutenin, a de-amidated glutenin, a prodynorphin, and a dynorphin.

The test results can advantageously be used to assist in differentiating gluten immune reactivity or sensitivity from celiac disease, especially where the wheat antigen is de—amidated, and the n antigen is selected from the group consisting of an a—gliadin—33-mer, oz-gliadinmer, din-lS-mer, an oa-gliadin-17—mer, and a glutenin—Zl-mer.

In certain s of the t invention, whole blood, blood sera, saliva or other samples from a human or other animal are tested for antibodies to one or more of a y-gliadin protein or a peptide thereof such as y—gliadin—15~mer, an m-gliadin protein or a peptide thereof such as (o- gliadin-l7-rner, wheat germ inin, a morphin, a glutenin protein or a peptide thereof such as glutenin—2l—mer, a de—amidated glutenin protein or a peptide thereof, a prodynorphin, and a dynorphin.

In certain aspects of the t invention, tests are conducted and/01‘ test results are analyzed for antibodies that assist in distinguishing gluten immune reactivity and/or ivity, silent or atypical celiac disease relative to patently symptomatic (classical) celiac disease, Crohn's disease and chronic immune activation. Test plates and kits of particular interest test for antibodies to at least three, five, seven or all of a—gliadin, y—gliadin, m-gliadin, glutenin, Wheat germ agglutinin, gluteomorphin, prodynorphins, transglutaminase, and gliadin—bound transglutaminase (gliadin-trans inase x).

In n aspects of the present invention, assays and assay kits of particular interest allow for testing IgA and/or IgG antibodies to one or more wheat antigens, a a—gliadin protein or one or more peptides thereof such as 01 -gliadinmer and/or at -g1iadinmer, a y-gliadin protein or one or more peptides thereof such as y—gliadin—lS-mer, an din protein or one or more peptides thereof such as co-gliadin-l7-mer, wheat germ agglutinin, an opioid peptide such as one or more of gluteomoxphin, prodynorphin and/0r dynorphin, a glutenin protein or one or more peptides thereof such as glutenin-Zl-mer, a de-amidated glutenin protein or one or more peptides thereof, a gliadin— lutaminase complex, or combinations thereof.

In certain aspects of the present invention, the detection of antibodies can be performed with an immunoassay, including, but not limited to, ELISA assay, RIA assay, latex agglutination, beads assay, proteomic assays, and other immunoassays known to one of ordinary skill in the art.

In one particular aspect the present invention proves a method of characterizing the antibody ition of a sample from an animal, comprising: HAS510070NZPR 303810428 measuring a first signal derived from binding of a first antibody of the sample to wholewheat n obtainable by combining water-soluble proteins and alcohol-soluble proteins extracted from wheat; measuring a second signal derived from binding of a second antibody of the sample to a gliadin antigen; and measuring a third signal derived from binding of a third dy of the sample to an n selected from the group consisting of: (1) a wheat germ inin; (2) gluteomorphin; (3) a glutenin; (4) a deamidated glutenin; (5) a orphin; and (6) a dynorphin, wherein the presence of the first, second, and third signals indicate the presence of the first, second and third antibodies in the sample, respectively.

In r particular aspect the present invention provides a method of generating an antibody profile that assists in differentiating between silent celiac e, gluten immune reactivity and sensitivity and gluten immune reactivity/sensitivity with autoimmunity, comprising: testing a sample from an animal for the presence of antibody against deamidated Į-gliadin 33-mer or transglutaminase-2; and testing the sample for the presence of antibody t deamidated in 21-mer, wherein indication of the presence of antibody against a designated antigen constitutes a positive test result for the designated antigen.

In another particular aspect the present ion provides a method of screening a sample from an animal for the presence of at least one of IgG and IgA dies directed to one or more wheat antigens, comprising ing a first, a second and a third signal, wherein the first signal is derived from binding of a first antibody of the sample to wheat antigen obtainable by combining water-soluble proteins and alcohol-soluble proteins extracted from wheat, the second signal is derived from g of a second antibody of the sample to a gliadin antigen; and the third signal is derived from binding of a third antibody of the sample to an antigen selected from the group consisting of: (1) a wheat germ inin; (2) gluteomorphin; (3) glutenin; (4) a de-aminated glutinin; (5) a prodynorphin; and (6) a dynorphin; wherein the presence of the first, second, and third signals indicate the presence of the first, second and third antibodies in the sample, respectively.

In another particular aspect the present invention provides a method of screening for the presence of IgG and/or IgA antibodies to wheat antigens in a sample from an animal, comprising measuring signals derived from binding of antibodies in the sample to HAS510070NZPR 303810428 (a) at least one of Į-gliadin and transglutaminase, and (b) at least one of whole-wheat antigen obtainable by combining water-soluble ns and alcohol-soluble proteins ted from wheat, wheat germ agglutinin, agglutinin, ω- and Ȗ-gliadins, glutenin, morphin, and gliadin-bound transglutaminase; wherein the presence of the signals indicate the ce of the respective antibodies in the sample.

In another particular aspect the present invention provides a test plate containing at least three bound test substances ed from the list consisting of whole-wheat antigen obtainable by combining water-soluble proteins and alcohol-soluble proteins extracted from wheat, Į-gliadin mer, Į-gliadinmer, Ȗ-gliadinmer, ω-gliadinmer, gluteninmer, gluteomorphin 16- mer, prodynorphin, gliadin-bound transglutaminase, and wheat germ agglutinin.

In another particular aspect the t invention provides a test kit comprising a test plate ning at least three bound test substances selected from the list consisting of whole-wheat antigen obtainable by combining water-soluble proteins and alcohol-soluble proteins extracted from wheat, Į-gliadinmer, Į-gliadinmer, Ȗ-gliadinmer, ω-gliadinmer, gluteninmer, gluteomorphin 16-mer, prodynorphin, gliadin-bound transglutaminase, and wheat germ agglutinin, and instructions for using the test plate to measure IgG and/or IgA antibodies.

W0 2012/100070 PCT/U82012/021892 Figure 4 is a diagram showing the layout of a sample microtiter plate by which IgG or IgA is measured with weekly negative and positive controls for quality control es (antigens or peptides are transparent).

Figure 5 is a diagram of an antibody testing ol for celiac disease using tTg and various antigens according to the prior art (17).

Figure 6 is a diagram of a testing protocol for ceiiac e using deamidated gliadin peptide according to the prior art (17).

Figure 7 is a diagram of a proposed testing protocol for celiac disease, gluten immune reactivity/sensitivity and autoimmunity using a repertoire of wheat ns and peptides according to certain aspects of the present invention.

DETAlLED DESCRIPTION OF THE DRAWINGS According to certain aspects of the present invention, antibodies are used as biomarkers to assist in diagnosing and distinguishing gluten immune reactivity and sensitivity, silent celiac disease, Crohn’s disease and other gut-related ogies as opposed to the classical celiac disease against an array of wheat antigens and peptides.

In certain aspects of the present invention, a bodily fluid is tested for immunoglobulin G (IgG) and/or immunoglobulin A (IgA) antibodies to one or more of a whole-wheat antigen; an a—gliadin protein or one or more peptides thereof; a y-gliadin protein or one or more peptides thereof; an co-gliadin protein or one or more peptides thereof; a in protein or one or more es thereof; one or more opioid peptides; a gliadin-transglutaminase complex (gliadin bound to transglutaminase); lutaminase; wheat germ agglutinin; and ations thereof.

In certain aspects of the present ion, the whole-wheat antigen is deamidated and can be prepared by ing water-soluble and alcohol-soluble wheat proteins. The a-gliadin n or one or more peptides thereof includes a—gliadin—33-mer and/or d-gliadinmer, with other d-gliadin peptides contemplated. The y-giiadin protein or one or more peptides thereof includes y—gliadin-lS-mer, with other y-gliadin peptides contemplated. The din protein or one or more es thereof includes 0)- gliadin—i7-mer, with other a) -g1iadin peptides contemplated. The glutenin protein or one or more peptides thereof includes in—ZI-mer, with other glutenin peptides contemplated. The one or more opioid peptides includes exorphin peptides including PCT/11520121021892 gluteomorphin, prodynorphin and/or dynorphin, with other exorphin peptides contemplated.

In certain s of the t ion, whole blood, blood serum/sera, saliva or other bodily fluid samples from a human or other animal are tested for antibodies to (a) a wheat antigen; (b) a gliadin n; and (c) one or more of a wheat germ agglutinin, a gluteomorphin, a glutenin, a de-aminated glutenin, a prodynorphin, and a dynorphin. Test results are considered ularly interesting where the wheat antigen is deamidated, and the gliadin antigen is selected from the group consisting of an o-gliadinmer, an no gliadin—l7-mer, a din-lS-mer, an m-gliadin-l7-mer, and a glutenin-Zl-mer. Test plates and kits can advantageously test for antigens to at least three, five, seven or all of ugliadin , y-gliadin, w-gliadin, glutenin, wheat germ agglutinin, gluteomorphin, prodynorphins, transglutaminase, and giiadin-bound transglutarninase.

In certain aspects of the present invention, assays and assay kits of particular interest allow for testing IgA and/or IgG antibodies to one or more wheat antigens, a ot- gliadin protein or one or more peptides thereof such as 0t~gliadin—33-mer and/or u—gliadin- l7-mer, a y-gliadin protein or one or more es thereof such as din-lS-mer, an em gliadin protein or one or more es thereof such as m-gliadin-l7-mer, wheat germ agglutinin, an opioid peptide such as one or more of morphin, prodynorphin and/or dynorphin, a giutenin protein or one or more peptides thereof such as glutenin-Zl-mer, a de-amidated glutenin protein or one or more peptides thereof, a gliadin—transglutaminase complex, or combinations thereof.

In certain s of the t invention, the detection of dies can be performed with an immunoassay, inciuding, but not limited to, ELISA assay, RIA assay, latex agglutination, beads assay, proteornic assays, and other immunoassays known to one of ordinary skill in the art.

Following are exemplary descriptions of assays, and their use and analysis with respect to some test patients. Although other materials and methods similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred method and materials are now described in the exemplary description of assays to further illustrate the present invention.

PCT/U52012/021892 EXAMPLE 1 ELISA Assay A. Materials And Methods - Plate and Sample ation: Wheat ns and peptides. A whole-wheat antigen was prepared by combining water-soluble and alcohol~soluble proteins. Different gliadin peptides including ot-gliadin 33-mer, 17-mer, y-gliadin lS-mer, co-gliadin l7-mer, gluteomorphin, prodynorphin, transglutaminases, and gliadin bound to transglutaminase, glutamic acid decarboxylase (GAD-65) HPLC grade were synthesized by Bio-Synthesis Inc. (Lewisville, TX). Wheat germ agglutinin (WGA) was sed from Sigma/Aldrich (Saint Louis, MO).

The antigens and peptides were dissolved in methanol at a concentration of 1.0 mg/mL, then diluted 1:100 in 0.1M carbonate-bicarbonate buffer, pH 9.5, and 50 pl were added to each well of a polystyrene flat-bottom ELISA plate.

The ELISA plates were incubated ght at 4°C and then washed three times with 200 pl Tris-buffered saline (TBS) containing 0.05% Tween 20 (pH 7.4). The non- specific binding of immunoglobulins was prevented by adding 200 mL of 2% bovine were washed and serum albumin (BSA) in TBS, and incubated overnight at 4°C. Plates after conducting quality control were kept at 4°C until used.

The enzyme conjugates included: Affinity Purified Antibody Phosphatase-labeled Goat anti-Human IgG on ImmunoResearch, Cat#109-055—008), and Affinity Purified Antibody Phosphatase-labeled Goat uman IgA (Jackson ImmunoResearch, 9-055~01 1).

Other additional reagents and als included in the method as r described herein, includes: Phosphate-Buffered Saline Powder (Sigma, Cat#P3813-10PAK), Bovine Serum Albumin (Biocell, 03-00), Sodium Azide (Sigma, Cat#S-2002), Tween 20 (Sigma, Cat#P1379-1000ML), ol (Sigma, Cat#GSSl6-500ML), Sodium Hydroxide (Sigma, Cat#S—5881), Magnesium Chloride (Sigma, Cat#8266), Diethanolamine (Sigma, Cat#D-8885), 1.0 N Hydrochloric Acid Solution (Sigma, Cat#I-I3162), 5mg Substrate Tablets: p—NPP (para-nitophenyl phosphate) , Cat#S-0942), and Distilled water (D.

H20).

The microwell plates were prepared and coated with the desired number of wheat- associated antigens and/or peptides. In the following case examples, 12 different wheat- PCT/U52012/021892 assocaited antigens and peptides were coated on the microwell plates. Calibrator and positive controls and diluted patient samples were added to the wells and autoantibodies recognizing different wheat antigens bind during the first incubation. After washing the wells to remove all unbound proteins, purified alkaline phosphatase labeled rabbit anti- human IgG/lgA unbound conjugate were removed by a r wash step.

Bound conjugate was visualized with paranitrophenyl phosphate (PNPP) substrate, which gives a yellow reaction product, the intensity of which is proportional to the concentration of autoantibody in the sample. Sodium hydroxide was added to each well to stop the on. The ity of color was read at 405 nm.

Plain red tops or red tiger tops (SST tubes) were used for specimen collection, this although in certain aspects, other specimen collection apparatus are plated for assay.

Blood samples were collected using aseptic ncture techniques and serum was obtained using standard procedures. In certain s, it is preferred that a minimum ml or more of about 50 pl of serum for the assay, which therefore corresponds to about 0.5 of blood.

B. Test Assay Procedure in diagnosing The analytical procedure for lgG and/or IgA antibody array to assist and detection of gluten immune reactivity and sensitivity, silent celiac disease, s e and other gut-related pathologies is now discussed. In some aspects, all ts test assay was commenced. The test were allowed to reach room temperature before the the desired number of coated wells or plates with the assay procedure includes preparing desired number and type of wheat-associated antigens and/or peptides. Once the added to microtiter wells are prepared, about 100 pl of 1:100 diluted control calibrator are multi-channel pipettor.

Rows A and B of the microtiter plate as shown in Figure 3 using a pl of 1:100 d patient’s test , here blood serum, was added to About 100 duplicate wells of rows C and D for Clinical Specimen 1, rows 13 and F for Clinical Specimen 2, and rows G and H for Clinical Specimen 3. clinical On a separate plate, the periodic ve and positive controls similar to specimens in duplicates were conducted, as shown in Figure 4.

PCT/U82012/021892 The plates were then incubated for 60 s at room temperature. After incubation, the wells were d and washed four times with PBS using an ELISA Washer. About 100 pl of optimally diluted alkaline phosphatase-labeled goat anti—human IgA was added to the IgA plate or about 100 pl of enzyme-labeled IgG was added to the IgG plate at optimal dilution.

The respective plates was then incubated for 30-60 s at room temperature.

About ten minutes before the conjugate-incubation ends, a ate solution was prepared by mixing 5 mg of a p-nitrophenyl phosphate tablet with 5 m1 of substrate buffer, which was mixed well until the tablet completely dissolved. Washing four times with PBS using the ELISA washer was repeated. Then, about 100 pl of substrate solution was added to each well. The plate was then incubated for 30 s at room temperature with the avoidance of any exposure to direct sunlight. The reaction was stopped by adding about 50 ul of 3 N NaOH. The color intensity of the wells were read using a microtiter plate reader at 405 nm t a blank well, with the absorbance values of the calibrators, controls and unknown samples being recorded.

C. Calculation of Results After the plate was read the plate at 405 nm to obtain the optical density values (OD4OS), the mean ODs of the negative controls, the mean 0135 of the positive controls and the mean ODs of each clinical specimen were divided by the mean ODs of calibrators on Rows A and B to obtain each Index Value (IV).

The Index Value (IV) for each antibody was calculated against the 12 different antigens by dividing the mean OD of each duplicate sample by the mean OD of the calibrator control value (for example, divide the mean OD of wells Cl and DI by the D2 by the mean OD of wells mean OD of wells A1 and Bl, the mean OD of wells CZ and A2 and B2, the mean OD of wells C3 and D3 by the mean OD of wells A3 and BB, etc.).

The results were then ed to the established reference ranges.

Mean OD of patients Index = __________.__.._......

Mean OD of calibrators PCT/U32012/021892 index ation For Wheat ns Cal 1 (OD) Cal 2 (OD) Sample 3 A (OD) Sample 3 B (OD) D. Interpretation of Results Examples of IgG and IgA antibody patterns of 3 patients with celiac e, 3 patients with gluten immune reactivity and sensitivity, and 3 patients with Crohn’s disease with overlapping gluten sensitivity are shown in Tables 2-7. Crohn's disease, also known as regional enteritis, is a type of inflammatory bowel disease having a strong genetic component. It is often described as an autoimmune disease, but others consider it be a disease of immune deficiency.

Data interpretation and laboratory differentiation between celiac disease and gluten immune reactivity/sensitivity/autoimmunity are shown in Table 8. In particular, it is now contemplated that celiac disease can be differentiated from silent celiac disease, gluten immune reactivity/sensitivity and/or gluten-related autoimmunity as follows: a. A serological n of silent celiac disease is indicated where the test results e positive results of IgA and/or IgG against any or combination of wheat proteins, oc-gliadin 33-mer, deamidated a-gliadin 33-mer, a-gliadin 25-mer, a-gliadin 18- deamidated in, mer, or-gliadin 17-mer, y-gliadin lS-mer, co—gliadin 17-mer, glutenin, gluteomorphin, prodynorphin and wheat germ inin, and negative results against tranglutaminase-Z, and at least one of IgA and IgG tests positive against at least one of transgiutaminase—3 and transglutaminase-6. b. A serological pattern of gluten immune reactivity and sensitivity is indicated where the test results e positive results for IgG and/or IgA against wheat ns, in particular native adin, y~gliadin, to-gliadin, in, deamidated glutenin, gluteomorphin, and wheat not germ agglutinin, but not dearnidated a~gliadin and transglutaminase-Z.

PCT/U52012/021892 c. A diagnosis related to gluten immune reactivity/sensitivity and autoimmunity is ted where the test results include a positive for IgG and/or IgA t wheat antigens, in particular native ougliadin, y-gliadin, w-gliadin, glutenin, deamidated glutenin, gluteomorphin, and wheat germ agglutinin, and positive results for tissue antigens or peptides any IgG, IgA or IgM to glutamic acid decarboxylase, cerebellar or other tissue antigens, and ve results for IgA to deamidated din, deamidated glutenin or t transglutaminase-Z, but positive IgA or IgG against transglutaminase-3 and/or transglutaminase-6.

Reportable ranges and reference ranges are show in Table 1, which may be updated from time to time. The reader will note that compared to established reference ranges at 2 rds above the mean, while IgA antibody t tTg and gliadin-tTg complex is highly positive (confirming the diagnosis of CD), the pattern and the strength of igG and IgA antibody varies from antigen to antigen. For example, while both IgG and IgA against wheat antigens in all three patients with celiac disease was 4-6 fold higher than reference ranges, when the IgG and IgA was ed against oc-gliadin 33-mer the of three IgG antibody level was significantly elevated in one and the IgA level in two out patients (Tables 2, 3).

When ed to patients with celiac disease, in patients with gluten immune reactivity and sensitivity none of the three patients showed a cant IgA reactivity against fig and gliadinvtTg complex, while the IgG and IgA antibodies against wheat antigens and in combination with one or more wheat peptides (a—gliadin, v- gliadin, 0)- gliadin, glutenin, gluteomorphin, prodynorphin and wheat germ agglutinin) was significantly elevated. The IgA immune reaction against wheat antigens and peptides ation with tTg and gliadin-tTg complex clearly distinguished between celiac antibodies are disease and gluten immune reactivity/sensitivity, in which IgG and/or IgA reactive against various wheat antigens and peptides but not against tTg and the gliadin- tTg complex (Tables 4, 5).

Tables 6 and 7 present the results of three ts with s disease. The shows that pattern of IgG and IgA antibodies against tTg and gliadin-tTg complex clearly these patients, in addition to Crohn’s disease, are also suffering from gluten immune reactivity/sensitivity, and possibly also celiac disease (Tables 6, 7).

W0 2012/100070 CASE STUDY EXAMPLES Four different case reports, the first on a patient with celiac disease, the second with gluten sensitivity, the third with gluten sensitivity and autoimmunity, and the fourth with gluten sensitivity overlapping with Crohn’s disease are shown below.

A. CASE REPORT #1: Diagnosis of Celiac Disease in the Elderly by the Use of IgA against Gliadin and Tissue Transglutaminase with Improvement on a Gluten-Free Diet A 76 ld man with longstanding dyspepsia, indigestion, ess and rapid weight loss was ed for gastrointestinal evaluation. Blood tests showed macrocytic anemia with low concentrations of foiate and vitamin B-12. The patient’s hemoglobin concentration was 79 g/L, albumin 32 g/L, and transglutaminase 212 ug/mL (normal range = 0-10 ug/mL. An urgent colonoscopy and al biopsy was performed, which yielded macrocospically normal results. At this level his IgG and IgA concentrations against gliadin and transglutaminase were checked using FDA-approved kits. Both IgG and IgA against or-gliadin were very high; against transglutaminase, lgA but not IgG was 3.8-fold higher than the reference In view of the IgA positivity against gliadin and range. transglutaminase and diagnosis of celiac disease he was rsed with 2 units of packed cells and started on both a gluten-free diet and 20 mg of prednisone daily. Six months later he had gained about 12 pounds and showed few GI symptoms. Because of this improvement the patient became committed to the gluten-free diet. One year after the first performance of IgG and IgA antibody testing against gliadin and lutaminase the repeat tests for these antibodies were negative, which is a further indication that disease management plus a gluten—free diet was mental in the treatment of this elderly patient with a silent celiac disease.

Discussion: According to Catassi et a1. (1, 11), celiac disease (CD) is one of the most common lifelong disorders in western countries. However, most cases of CD remain undiagnosed mostly due to the poor awareness of the primary care physician regarding this important affliction (Catassi C, et al., Am J Gastroenterol, 102:1454—1460, 2007). Celiac e is perceived as presenting GI ms accompanied by maiabsorption. But many patients with celiac disease do not present GI symptoms. These individuals may have silent or al celiac disease, and the condition may present with iron deficiency, , sed liver s, osteoporosis or neurological symptoms (12). As used herein, the term “atypical celiac e” refers to celiac disease in patients who have only subtle symptoms, and the term “silent celiac disease” refers to celiac disease in ts who are asymptomatic.

The increasing recognition of celiac disease is attributed to the use of new serological assays with higher sensitivity and specificity. Until recently celiac e was ectly perceived as being uncommon and detected mainly during infancy or childhood. r, it is now recognized that most cases of CD occur in adults 40-60 lab test results and other years old. Patients in this age group may present their symptoms, examination signs in atypical fashion. In fact, according to a very recent publication, less than one in seven patients is correctly diagnosed with CD (13). uently, as this case shows, if an adult t presents with symptoms and signs suggesting malabsorption, testing for IgA antibody against gliadin and transglutaminase should be considered. If the test results are positive, celiac diseases should then be made a part of the differential diagnosis, based on which a gluten-free diet should be recommended. If the gluten-free diet should produce an ement in symptoms, the patient should commit to the diet regardless of age.

B. CASE REPORT #2: Gluten Immune Reactivity and ivity induced by a Combination of Anesthetics, Antibiotics and Pain Medication A 46 year-old woman was given her yearly checkup by her internist. Based on medical examination and a normal CBC, chemistry including liver enzymes, and an autoimmune profile, she was classified as a healthy person. Gluten antibodies were not measured at that time. A few months later she went to her dentist for a root canal, bone graft and preparation for dental implantation. During five ent visits over ten days treated with anesthetic material (mepivacaine), otics and painkillers. Four months later the dental implant procedure was completed after local anesthesia with lidocaine with subsequent prescription of antibiotic (amoxicillin) and painkillers. Four hours later she developed a severe allergic reaction with localized edema, in particular lips and periorbital with a area swelling. The t became agitated and exhibited generalized itching, particularly her face, hands and feet. Tightness of the chest with wheezing and difficulty in breathing was an indication of allergic reaction to one or more of the medicines used. She was immediately treated with 0.01 mL per kg of body weight of adrenaline, intramuscularly supplemented by antihistamine treatment. However, while PCT/U52012/021892 the allergic reaction was controlled, the patient developed severe vomiting and diarrhea with severe nal pain, which lasted for 8 days. Two weeks later, while the diarrhea had ameliorated, the patient continued to complain about bloating and abdominal fort with irritable bowel-like syndrome. She was referred to a GI specialist who detected nothing of note upon a thorough examination. The ility of gluten sensitivity was then considered, and the patient was tested for HLA typing and IgG and IgA anti- gliadin and anti-transgiutarninase antibodies. Immunological tests showed these s: IgG anti-gliadin 6.8 U/mL (normal range <20 U/mL); IgA anti-gliadin 4.1 U/mL (normal range <20 U/mL); IgG Tg 2.1 U/mL (normal range <6 U/mL); IgA anti-tTg 1.2 U/mL (normal range <4 U/mL); and negative for HLA DQZ and DQS. Based on these findings gluten sensitivity and celiac e were excluded, and it was concluded that the patient was suffering from psychogenic or idiosyncratic reaction associated with reaction to the anesthetic and its synergistic effect with the antibiotics. Ninety days later upon follow-up visit the patient was still complaining about bloating and abdominal pain, particularly 1-3 hours after each meal. Repeat testing was ordered, and both a basic test and a comprehensive test was ordered for anti-gliadin and -tTg IgG and IgA along with ASCA and p-ANCA IgG, which are the suggested tests for suspected Crohn’s disease and ulcerative colitis. Interestingly, almost 100 days after the first GI discomfort, while ASCA and p-ANCA were completely within the normal range, both the IgG and IgA against = 54 2O gliadin were 4 to 9-fold above the nce range (gliadin IgG = 79 U/mL; IgA U/mL). However, IgG and IgA antibodies against tTg were within the normal range. addition, the IgG and IgA antibody testing was performed against an array of wheat, gliadin, glutenin, wheat germ agglutinin, n-tTg complex and fig ns. IgG was detected against 8 out of 12 tested antigens and IgA against 6 out of 12 tested antigens at 2-7 fold higher than established reference ranges. Both IgG and IgA against fig gliadin «tTg complex were negative (see Tables 4 and 5, Sample #1). These results along with the positivity of the basic IgG and IgA test t gliadin but not against transglutaminase showed that the patient, due to allergic reaction to environmental factors, had lost nce to wheat antigens and had developed gluten sensitivity but not celiac disease. Despite the e of elevation in IgG and IgA levels against tTg, due to continuous GI discomfort and the elevated IgG and IgA t gliadin, a gluten-free diet was recommended, and a dietitian advised the patient to take tics and go on restricted diet free of glutens and also of lectins, since the WGA level was also elevated for both lgG and lgA. Six months afier the introduction of the diet and the probiotics, the patient’s GI discomfort had subsided and she was back to normal health.

Discussion: The term gluten sensitivity refers to a state of heightened immunological responsiveness to gluten as indicated by the ion of IgG, IgA or both against n but not against transglutarninase (14). Gluten sensitivity begins with the loss of mucosal immune tolerance to wheat antigens and peptides due to environmental factors affecting the mucosal immune tasis.

In this case gluten ivity was continued based on G1 symptoms and immunological testing, in particular IgG and IgA against gliadin and its associated proteins and peptides almost 100 days after the triggering s had affected her state of immunological tolerance to wheat and associated antigens. It seems that in this patient the synergistic effects of anesthetics, antibiotics and painkillers resulted in ulation of her mucosal immune system, followed by a breakdown in immunological tolerance to wheat and other dietary proteins and peptides. This, ly in combination with the effect of environmental factors on the activity of the digestive enzymes, resulted in the induction of the opening of tight junctions and the entry of undigested wheat proteins and peptides into the submucosa, lymph nodes, and the circulation. These antigens were subsequently presented by antigen—presenting cells to T cells and B cells. During this T cells, leading to B—cell process gliadin-specific B cells are assisted by n-specific clonal expansion and the release of lgG and lgA antibodies to gliadin and associated proteins and peptides, which in this case was detected about 100 days after the original traumatic experience.

It is concluded herein that ing for gluten sensitivity in patients with GI discomfort associated with the use of etics and antibiotics may be easily and cost- effectively undertaken by measuring circulating IgG and IgA against gliadin and associated proteins and peptides. Failure to do so may not only e the patient of an accurate diagnosis and the proper treatment by implementation of a gluten~free diet, but side s. may also result in unnecessary medical interventions with their associated C. CASE REPORT #3: Gluten Immune Reactivity, Sensitivity and Autoimmunity Here, a case report is described in which the al tation led to an erroneous diagnosis of irritable bowel syndrome, resulting in incorrect medical PCT/U82012/021892 ention. The correct diagnosis of gluten immune reactivity and sensitivity was made after years of mistreatment.

A 49 year-old woman with abdominal pain, constipation, acid reflux and headache was examined by an internist. Investigation revealed normal CBC with hemoglobin of .8 g/dl and normal chemistry profile including liver enzyme. Over several visits detailed biochemical and immunological profiles ing ANA, rheumatoid factor, T3, T4, and TSH levels were performed, all testing within the normal range. After repeated complaints about GI discomfort, the patient was referred for GI evaluation. Both endoscopy and H. pylori test results were normal. The patient was diagnosed with irritable bowel syndrome and put on B-blockers and , which moderately improved her symptomatologies. Four years later, however, in addition to the old GI symptoms and headache, she presented symptoms of malaise, d vision and facial rash. She was intermittently sleepy and irritable, and experienced breathing problems. Further lab tests revealed her hemoglobin was 9.7 g/dl with MCV of 72 fL, a raised erythrocyte sedimentation rate (46 mm/ist hour), ANA of 1:80 (normal range <40), mild elevation in IgA smooth muscle dy, double-stranded DNA and extractable nuclear antibodies were negative. Based on the available evidence, a diagnosis of systemic lupus erythematosus (SLE) was made by a rheumatologist, and treatment with steroids was commenced. There was some improvement in her overall state but her hemoglobin level continued to be low, while her ESR fluctuated. Two years later she developed lty in passing urine accompanied by tingling and sensory disturbance in her trunk and legs, which led to her being referred to a neurologist. Close oning revealed a band-like sensation in the trunk and reduced visual acuity (8/46 in the right eye, 8/23 in the left eye) with minimal eye pain, but normal eye movement. Lab investigation came up with low hemoglobin, abnormal MCV, and low serum ferritin at 14 ug/L (normal range 10-150 ug/L), which ed iron deficiency. MRI scan of the brain showed extensive white detected matter abnormalities not typical of multiple sis, but no alities were in CSF examination. While blood and CSF examination showed no evidence of ial and viral infection including syphilis, mycobacteria, ia, EBV, CMV, HTLV, and view of Herpes Type~6, visual evoked potentials showed delay in both optic nerves. In these abnormalities, and since tests for gluten sensitivity had not been performed during the r investigations, the possibility of gluten sensitivity was considered. A comprehensive IgG and IgA panel was ordered against a repertoire of wheat proteins and PCT/U82012/021892 peptides, as well as against tTg and s tissue antigens. This hensive gluten sensitivity and immune reactivity screen revealed IgG t wheat antigens, a-gliadin 33- and , y— and (o-gliadin, glutenin, gluteomorphin, prodynorphin, gliadin—tTg complex, wheat germ agglutinin, and glutamic acid decarboxylase 65 (GAD-65). IgA antibodies were detected against wheat antigens and wheat germ agglutinin (see Tables 4 and 5, Sample #3). Interestingly, both lgA and IgG tested against tTg were within the normal range.

Furthermore, antibodies against ganglioside, cerebellar, synapsin, myelin basic protein, collagen, thyroglobulin and thyroid peroxidase were , and all were 2-4 fold above the reference range. Upper GI endoscopy and biopsy revealed normal histology and intraepithelial lymphocytes. Overall the patient was diagnosed as having gluten sensitivity with its associated autoimmunities, including gluten ataxia, headache, white matter abnormalities, and neuromyelitis optica. A five-day course of intravenous prednisolone was implemented, and gradually the sensory, motor and visual of IgG and some IgA symptoms improved. In addition, based on the very high levels dies against a repertoire of wheat ns and peptides, a gluten-free diet was introduced, and 12 weeks later marked improvement was observed in the patient’s clinical symptomatology. She continued the 100% gluten~free diet under the observation of a of the diet dietitian, and the d treatment was stopped. Six months after introduction dy tests against wheat ns, peptides, and human tissue were repeated; more than 60% reduction in antibody levels was observed, and the patient became almost asymptomatic.

Discussion: From this data I have concluded that a patient may suffer from gluten flat erosive immune reactivity and ivity without having abnormal tissue histology or gastritis and antibody against tTg based on which a diagnosis of celiac disease is normally made. If ts with gluten sensitivity and immune reactivity are not detected in time based on the proper lab tests, in particular IgG and IgA antibodies against a repertoire into wheat proteins and peptides, ts’ symptomatologies may mislead many clinicians other treating their patients for lupus, MS-like syndrome, neuromyeltitis optica, and many autoimmune disorders. Therefore, measurement of IgG and IgA antibodies against a repertoire of wheat antigens and peptides is recommended for patients with signs and symptoms of autoimmunities be so that intervention with a gluten~free diet will instrumental in reversing the autoimmune conditions associated with gluten immune W0 2012/100070 PCT/U52012/021892 reactivity and sensitivity. Otherwise, untreated and/or mistreated, the patient will develop multiple autoimmune disorders.

D. CASE REPORT #4: Gluten Immune Reactivity and Sensitivity Overlapping with Crohn’s Disease Crohn’s disease is an inflammatory disorder that often emerges during the second or third decade of life, affecting the terminal ileum in more than two-thirds of patients (15). A combination of genetic and environmental factors, including a shift in gut flora and ctional responses against them, is believed to lead to dysregulated immunity, altered intestinal barrier function, and possibly autoimmunity (16).

Here, a 32 year-old man presented with gastrointestinal fort and diarrhea 2- 3 times per month. Laboratory results including chemistry panel, CBC, iron, in, errin, vitamin B-12, thyroid fimction, and urine analysis were within the median level of the normal range. Upon the second visit and continuation of GI symptoms he was referred to a GI specialist who ordered additional lab ations. These tests were microbiological tion of the stool and blood tests for antibodies against H. pylori, Saccharomyces and gliadin. Stool testing with respect to the detection of ella, Shigella, Yersinia, Campylobacter, enteropathogenic and enterohemorrhagic E. coli or Clostridium difficile came out negative. Regarding antibody examinations in the blood, IgG against H. pylori and IgA against Saccharomyces and gliadin were negative, but IgG against gliadin was moderately elevated at 59 U/rnL (normal values = <20 U/mL). The IgG antibody ions were ered non-specific or protective, and the patient was Three years put on painkillers and sent home with no diagnosis of any specific disorder. later after seeing the frequency of the watery ea increase to 3-5 times daily and losing 12 pounds of his body weight in the last two months, the patient went to another GI list for a second opinion. Gastric and duodenal biopsies and opy were performed. While the endoscopy of the upper GI tract revealed gastritis of the antrum, histologically, gastric and duodenal biopsy turned out to be negative. se absorption test was performed; the resulting value of 1.89 g/Sh in urine was suggestive of malabsorption. Immunoserologically ANA titers were below 1:40, p-ANCA and c-ANCA at 85 U/mL were negative, but the IgA anti-Saccharomyces antigen (ASCA) was positive (normal = <10 U/mL). Based on the increased ncy of watery diarrhea, abnormal D- xylose absorption, and positive IgA anti-ASCA, the diagnosis of Crohn’s disease was made. A therapeutical trial using cholestyramine was ted but the frequency of the PCT/U52012/021892 diarrhea ed unchanged. In addition the t was treated with 230 mg of methylprednisolone, and 2 x 1000 mg of mesalazine. Two years after this treatment the patient developed entero-enteric fistulae in the terminal ileum with sigmoid affection.

Afier admission to the hospital, ileocolectomy was performed and 22 cm of the ileum was resected. Upon his e ion maintenance with 3 x 500 mg of mesalazine was implemented.

For eight years following this treatment the patient continued to suffer from increasing frequency of watery diarrhea and lost an additional 14 pounds. During this period several additional ent attempts were made using aspirin, loperamide, and budesonide, unfortunately without significant al ement. Furthermore, the t was losing more weight on a monthly basis. A complete review of the medical history revealed the fact that almost thirteen years earlier, n IgG antibody had been found to be elevated, which was considered normal at the time. Since all classical treatments for Crohn’s disease had failed to improve the clinical picture over all the years, was a comprehensive test for the assessment of gluten immune reactivity and sensitivity ordered. This included IgG and IgA against wheat, native and deamidated a-gliadin peptides, y-gliadin, cc-gliadin, glutenin, gluteomorphin, prodynorphin, gliadin-tTg complex, transglutaminase, wheat germ agglutinin, and GAD-65.

Results ed in Tables 6 and 7, Sample #3 show that the patient had a significant elevation of IgG antibodies against ll out of 12 tested antigens, and IgA antibodies t wheat, a-gliadin 33-mer, m-gliadin, prodynorphin, wheat germ agglutinin and GAD-65 were detected at 2—5 fold above the normal range. Based on these results, in addition to Crohn’s disease a sis of gluten sensitivity was also made. A diet consisting of rice, , and other gluten-free/yeast-free foods was commenced immediately, which led after six weeks to a complete cessation of diarrhea. Upon uation of the gluten-free diet, not only did stool consistency become normal but the patient also started g weight. On follow-up one year later the patient was back to a normal state and had regained more than 80% of his lost weight.

Discussion: This case reports on the association of Crohn’s disease with gluten sensitivity but not with celiac disease. Based on the impressive clinical response to the gluten—free diet plus the detection of IgG and IgA antibodies against various wheat antigens, and upon re-evaluation of the IgG antibody level detected 14 years earlier, the W0 2012/100070 PCT/U52012/021892 diagnosis of Crohn’s disease with secondary malabsorption and gluten sensitivity was finally established. Since IgG antibodies against gliadin but not transglutaminase were detected, it can be argued that in this patient the disease initiated with gluten sensitivity and not Crohn’s disease. The initial diagnosis of Crohn’s disease was made despite the fact that a demonstration of duration exposure to gluten and risk of mune disorders was published in 1999 (Ventura A et al., Gastroenterol, 117: 0, 1999); unfortunately, this was ignored.

It is contemplated herein that continuous exposure to enviromncntal s such as wheat antigen induced inflammation for a prolonged period of time, ing in inflammatory bowel disease or Crohn’s disease.

Conclusions Regarding Case Reports The foregoing case studies show the importance of proper laboratory testing for confirming diagnoses of celiac disease, gluten immune reactivity/sensitivity, and autoimmunity. They show that years of ous testing and misdiagnoses can lead to years of suffering. It is vital to get the most accurate information and the most accurate diagnosis, to distinguish between one condition and another. Figures 5 and 6 show a summary of the current state of testing as well as a proposed future direction for more accuracy in the diagnosis of celiac disease as proposed by Volta et a1. (17). Figure 7 summarizes an inventive protocol according to certain aspects of the present invention, and proposes testing against a repertoire of wheat antigens and peptides so as to provide the most accurate ation and confirmation of celiac disease, Crohn’s disease, gluten immune reactivity/sensitivity and autoimmunity. it should be apparent to those skilled in the art that many more modifications s those already bed are possible without departing from the inventive concepts . The inventive subject matter, therefore, is not to be cted except in the scope of the appended claims. Moreover, in interpreting both the specification and the claims, all terms should be interpreted in the broadest le manner tent with the context. In particular, the terms “comprises" and “comprising” should be interpreted as referring to elements, components, or steps in a non-exclusive manner, indicating that the referenced elements, components, or steps may be present, or utilized, or ed with other elements, ents, or steps that are not expressly referenced. Where the specification claims refers to at least one of ing selected from the group consisting of A, B, C and N, the text should be interpreted as requiring only one element from the group, not A plus N, or B plus N, etc.

The reference to any prior art in the specification is not, and should not be taken as, an acknowledgement or any form of suggestion that the prior art forms part of the common general dge in New Zealand.

REFERENCES l. Catassi C, Fasano A. Celiac disease. Curr Opin Gastroenterol, 24: 687- 691, 2008. 2. Anderson LA, an SA, Watson RG, et al. Malignancy and mortality in a population-based cohort of patients with coeliao disease or ‘gluten sensitivity.’ World J Gastroenterol, 13: 146—151, 2007. 3. Tanabe 8. Analysis of food allergen ures and development of foods for allergic patients. Biosci Biotechnol Biochem, 72: 649-659, 2008. 4. Chin RL, Latov N, Green PHR, et a1. Neurological complications of ccliac disease. J Clin Neuromusc Dis, 5: 129-137, 2004.

S. Sapone A, Lammers KM, Mozzarella G, et al. Differential mucosa] IL—l7 expression in two gliadiminduoed disorders: gluten sensitivity and the autoimmune enteropathy ccliac disease. Int Arch Allergy Immzmol. 152: 75-80, 2010. 6. Sapone A, s KM, Casolaro V, et a1. Divergence of gut permeability and mucosal immune gene expression in two gluten-associated conditions: eeliac disease and gluten sensitivity. BMC Med. 9: 23, 2011. 7. Vojdani A, O’Bryan T, mann GB. The immunology of immediate and delayed hypersensitivity reaction to gluten. Eur J Inflamm. 6(1): 1 ~l 0, 2008. 8. Sher DB, Bamilai 0, Ram M, et al. Gluten sensitivity in multiple sis: experimental myth or clinical truth? Ann NYAcad Sci, 1173: 343-349, 2009. 9. Vojdani A, n T, Green JA, et al. Immune response to dietary proteins, n and cerebellar peptides in children with autism. Nutr Neurosci. 7(3):15l- 161, 2004.

. Hadjivassiiiou M, Grunewald RA, Lawden M, et a1. Headache and CNS white matter abnormalities associated with gluten sensitivity. Neurol, 56: 385-388, 2001. 11. Catassi C, et a1. ion of Celiao disease in primary care: a multicenter ease-finding study in North a. Am J Gastroemeral, 102:1454—1460, 2007 . 12. Sanders DS, et a1. Antibody negative coeiiac disease presenting in elderly people—an easily missed diagnosis. BMJ, 330: 775-776, 2005. 13. Matthias 'l‘, Neidhofer S, Pfeiffer S, et a1. Novel trends in celiac disease.

Cell. Mol. l. 8: 121-125, 20} 1. 14. Jacob S et al. Gluten sensitivity and neuromyelitis optica: Two case reports.

J Neurol Neurosurg Psychiany, 76: 10234030, 2005.

. Egan CE, et a1. y between intraepitheliai lymphocytes and lamina propria T cells drives intestinal inflammation during ion. Mucosal Immunol, 4: 658- 670, 201 i. 16. Kaser A et a1. inflammatory Bowel Disease. Annu Rev Immunol, 28: 573- 621, 2010. 17. Volta U and aci V. Celiac e: diagnostic criteria in progress.

Cell Mol Immunol, 8: 96—102, 201 1. 18. Vojdani A. 2011. The characterization of the repertoire of wheat antigens and peptides involved in the humoral immune responses in patients with gluten sensitivity and Crohn’s disease. ISRN Ailergy. Article ID 950104, 1-12.

HAS510070NZPR 303810425

Claims (32)

1. A method of characterizing the antibody composition of a sample from an animal, comprising: measuring a first signal derived from binding of a first antibody of the sample to wheat antigen obtainable by combining water-soluble proteins and alcohol-soluble proteins extracted from wheat; measuring a second signal derived from binding of a second antibody of the sample to a gliadin antigen; and measuring a third signal derived from binding of a third antibody of the sample to an antigen selected from the group consisting of: (1) a wheat germ agglutinin; (2) morphin; (3) a glutenin; (4) a ated glutenin; (5) a prodynorphin; and (6) a dynorphin, wherein the presence of the first, , and third signals indicate the presence of the first, second and third antibodies in the sample, respectively.

2. The method of claim 1, wherein the sample comprises blood serum.

3. The method of claim 1, n the sample comprises .

4. The method of any one of claims 1 to 3, wherein the n antigen is selected from the group consisting of an Į-gliadinmer, an Į-gliadinmer, a ɶ-gliadinmer, an ωgliadinmer.

5. The method of any one of claims 1 to 4 wherein the glutenin antigen is a gluteninmer.

6. The method of claim 4 or claim 5, wherein at least one of the gliadin and/or glutenin antigens is deamidated.

7. The method of any one of claims 1 to 6, wherein the animal is a human being. HAS510070NZPR 303810425

8. A method of generating an antibody profile that assists in differentiating between silent celiac disease, gluten immune reactivity and ivity and gluten immune reactivity/sensitivity with autoimmunity, comprising: testing a sample from an animal for the presence of antibody against deamidated Į-gliadin 33-mer or transglutaminase-2; and testing the sample for the presence of antibody against ated glutenin 21-mer, wherein indication of the presence of antibody against a ated n constitutes a positive test result for the designated antigen.

9. The method of claim 8 r comprising determining classes of antibodies present in the sample, wherein an antibody profile comprising the following assists in entiating silent celiac disease from gluten immune reactivity and sensitivity, and from gluten immune reactivity/sensitivity with autoimmunity: at least one of IgA and IgG test ve against at least one of Į-gliadin 33-mer, deamidated Į-gliadin 33-mer, Į-gliadin 25-mer, Į-gliadin 18-mer, Į-gliadin 17- mer, ɶ-gliadin 15-mer, din 17-mer, glutenin, deamidated glutenin, gluteomorphin, prodynorphin and wheat germ agglutinin; both IgA and IgG test negative against tranglutaminase-2; and at least one of IgA and IgG tests positive against at least one of transglutaminase-3 and transglutaminase-6.

10. The method of claim 8 further comprising determining classes of antibodies present in the sample, wherein an antibody profile comprising the following assists in differentiating gluten immune reactivity and sensitivity from silent celiac disease, and from gluten immune reactivity/sensitivity with munity: at least one of IgA and IgG test ve against at least one of Į-gliadin, din, ωgliadin , glutenin, deamidated glutenin, gluteomorphin, and wheat germ agglutinin; and both IgA and IgG test negative against deamidated Į-gliadin and/or transglutaminase-2.

11. The method of claim 8 further comprising determining classes of antibodies present in the sample, wherein an antibody profile comprising the ing assists in differentiating gluten HAS510070NZPR 303810425 immune reactivity/sensitivity with autoimmunity from silent celiac disease, and from gluten immune reactivity and sensitivity: at least one of IgA and IgG test positive against at least one of native Į-gliadin, in , ω-gliadin, glutenin, deamidated glutenin, gluteomorphin, and wheat germ agglutinin; at least one of IgG, IgA and IgM test positive to at least one of glutamic acid decarboxylase and cerebellar tissue; IgA tests ve against deamidated Į-gliadin, deamidated glutenin or transglutaminase-2; and IgA or IgG test positive against transglutiminase-3 and/or transglutaminase-6.

12. A method of screening a sample from an animal for the presence of at least one of IgG and IgA antibodies directed to one or more wheat antigens, comprising ing a first, a second and a third signal, wherein the first signal is derived from g of a first antibody of the sample to whole-wheat antigen obtainable by combining soluble proteins and alcohol-soluble proteins extracted from wheat, the second signal is derived from binding of a second antibody of the sample to a gliadin antigen; and the third signal is derived from binding of a third antibody of the sample to an antigen ed from the group consisting of: (1) a wheat germ agglutinin; (2) gluteomorphin; (3) glutenin; (4) a de-aminated glutinin; (5) a prodynorphin; and (6) a dynorphin; wherein the presence of the first, second, and third signals indicate the presence of the first, second and third antibodies in the sample, tively.

13. The method of claim 12 wherein the IgG and/or IgA antibodies to whole-wheat antigen bind to a wheat peptide and wherein the IgG and/or IgA antibodies to the n antigen bind to a gliadin peptide.

14. The method of claim 12, wherein the IgG and/or IgA antibodies to whole-wheat antigen bind to alcohol-soluble and/or water-soluble ns of wheat, and the gliadin antigen is HAS510070NZPR 303810425 selected from the group consisting of Į-gliadinmer, Į-gliadinmer, ɶ-gliadinmer, ω-gliadinmer.

15. The method of any one of claims 12 to 14 wherein the glutenin antigen is glutenin mer.

16. The method of any one of claims 12 to 15, further comprising using the test results to assist in ascertaining whether the animal has celiac disease.

17. The method of any one of claims 12 to 15, further comprising using the test results to assist in distinguishing whether the animal has silent or al celiac disease, as opposed to patently symptomatic celiac disease.

18. The method of any one of claims 12 to 15, further sing using the test results to assist in ascertaining whether the animal has at least one of gluten immune reactivity/ ivity with autoimmunity.

19. The method of any one of claims 12 to 15 further comprising using the test results to assist in ascertaining whether the animal has at least one of Crohn’s disease and chronic immune activation.

20. The method of any one of claims 16 to 19, further comprising testing the sample for at least five of IgG and/or IgA dies to Į-gliadin, ɶ-gliadin, ω-gliadin, glutenin, wheat germ agglutinin, morphin, prodynorphins, transglutaminase, and gliadin-bound transglutaminase.

21. A method of screening for the presence of IgG and/or IgA antibodies to wheat antigens in a sample from an animal, comprising measuring signals derived from binding of dies in the sample to (a) at least one of Į-gliadin and transglutaminase, and HAS510070NZPR 303810425 (b) at least one of whole-wheat n obtainable by combining water-soluble proteins and alcohol-soluble proteins extracted from wheat, wheat germ agglutinin, ω- and Ȗ -gliadins, glutenin, gluteomorphin, and n-bound transglutaminase; n the presence of the signals indicate the presence of the respective antibodies in the sample.

22. A test plate containing at least three bound test substances selected from the list consisting of whole-wheat antigen obtainable by combining water-soluble proteins and alcohol-soluble proteins extracted from wheat, Į-gliadinmer, Į-gliadinmer, dinmer, ωgliadin- 17-mer, gluteninmer, gluteomorphin 16-mer, prodynorphin, gliadin-bound transglutaminase, and wheat germ agglutinin.

23. The test plate of claim 22, containing at least five of the bound test substances.

24. The test plate of claim 22, containing at least seven of the bound test substances.

25. The test plate of claim 22, containing each of the bound test substances.

26. A test kit comprising the test plate of claim 22, and instructions for using the test plate to measure IgG and/or IgA antibodies.

27. A method as claimed in claim 1, substantially as hereinbefore described with particular reference to the examples and/or figures.

28. A method as claimed in claim 8, ntially as hereinbefore described with particular reference to the examples and/or figures.

29. A method as claimed in claim 12, substantially as hereinbefore bed with particular reference to the examples and/or figures. HAS510070NZPR 303810425

30. A method as claimed in claim 21, substantially as hereinbefore described with particular reference to the examples and/or figures.

31. A test plate as claimed in claim 22, substantially as hereinbefore described with ular reference to the examples and/or figures.

32. A test kit as claimed in claim 26, substantially as hereinbefore described with ular reference to the examples and/or figures. REPLACEMENT SHEET