US20120190571A1 - Methods and apparatus for detection of gluten sensitivity, and its differentiation from celiac disease - Google Patents

Methods and apparatus for detection of gluten sensitivity, and its differentiation from celiac disease Download PDF

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US20120190571A1
US20120190571A1 US13/354,119 US201213354119A US2012190571A1 US 20120190571 A1 US20120190571 A1 US 20120190571A1 US 201213354119 A US201213354119 A US 201213354119A US 2012190571 A1 US2012190571 A1 US 2012190571A1
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gliadin
mer
glutenin
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wheat
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Aristo Vojdani
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CYREX LABORATORIES LLC
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Immunosciences Lab Inc
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/06—Gastro-intestinal diseases
    • G01N2800/065—Bowel diseases, e.g. Crohn, ulcerative colitis, IBS
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/24—Immunology or allergic disorders

Definitions

  • the present invention relates to methods and kits for aid in diagnosis of gut-related diseases and pathologies, including at least gluten immune reactivity and sensitivity, silent celiac disease, and Crohn's disease.
  • CD Celiac disease
  • Markers for confirming a diagnosis of this disorder are IgA against native, deamidated gliadin peptides and IgA anti-tissue transglutaminase (tTg) autoantibody.
  • GS gluten sensitivity
  • symptoms in GS may resemble some of the gastrointestinal symptoms that are associated with CD or wheat allergy, but it is emphasized that objective diagnostic tests for gluten sensitivity are currently missing (5, 6).
  • TLR1, 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 IFN-g, IL-21 and IL-17A were expressed in mucosal tissue in CD but not GS. They believed that measurements of toll-like receptors and IFN- ⁇ , IL-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 hypersensitivity to gluten is an antibody-(IgG.
  • IgA T-cell-mediated reaction
  • celiac disease gluten sensitivity with enteropathy (7)
  • 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 development of gluten sensitivity (7).
  • patients refer to humans under the care of a health care professional. More broadly, however, the novel testing protocols and analyses disclosed herein could be applied to non-patient humans, and any other animal that could suffer from celiac disease, gluten sensitivity, and gut-related autoimmunities.
  • FIG. 1 A comparison between celiac disease and gluten immune reactivity/sensitivity is shown in FIG. 1 .
  • HLA DQ2/DQ8 a negative genetic makeup
  • HLA DQ2/DQ8 + the other with positive
  • the result can be a breakdown of mucosal immune tolerance in both children.
  • the IgG and IgA antibodies against gliadin along with biomarkers of inflammation can activate tTg, induce damage to the villi, and result in villous atrophy.
  • Deamidation of a specific gliadin peptide leads to the formation of a complex between it and the tTg; the presentation of this complex by antigen-presenting cells to T cells and B cells results in IgA or IgG production against 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 cancer.
  • the wheat antigens and reacting antibodies form an unholy alliance of immune complexes, resulting in severe gluten immune reactivity and sensitivity.
  • 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.
  • a gluten-free diet usually is only recommended for those who meet the criteria for a diagnosis of CD, not of gluten immune reactivity and sensitivity. Unfortunately, that leaves many gluten-sensitive people suffering unnecessarily with very serious symptoms that put them at risk for complications, conditions that might be resolved with a gluten-free diet, if they only knew.
  • 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 sensitivity, silent celiac disease, Crohn's disease and other gut-related pathologies.
  • whole blood, blood sera, saliva or other samples from a human or other animal are tested for antibodies to (a) a wheat antigen; (b) a gliadin antigen; and (c) one or more of a wheat germ agglutinin, a gluteomorphin, a glutenin, a de-amidated glutenin, a prodynorphin, and a dynorphin.
  • 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 gliadin antigen is selected from the group consisting of an ⁇ -gliadin-33-mer, an ⁇ -gliadin-17-mer, a ⁇ -gliadin-15-mer, an ⁇ -gliadin-17-mer, and a glutenin-21-mer.
  • whole blood, blood sera, saliva or other samples from a human or other animal are tested for antibodies to one or more of a ⁇ -gliadin protein or a peptide thereof such as ⁇ -gliadin-15-mer, an ⁇ -gliadin protein or a peptide thereof such as ⁇ -gliadin-17-mer, wheat germ agglutinin, a gluteomorphin, a glutenin protein or a peptide thereof such as glutenin-21-mer, a de-amidated glutenin protein or a peptide thereof, a prodynorphin, and a dynorphin.
  • a ⁇ -gliadin protein or a peptide thereof such as ⁇ -gliadin-15-mer
  • an ⁇ -gliadin protein or a peptide thereof such as ⁇ -gliadin-17-mer
  • wheat germ agglutinin a gluteomorphin
  • a glutenin protein or a peptide thereof such as glutenin-21-mer
  • tests are conducted and/or test results are analyzed for antibodies that assist in distinguishing gluten immune reactivity and/or sensitivity, 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 ⁇ -gliadin, ⁇ -gliadin, ⁇ -gliadin, glutenin, wheat germ agglutinin, gluteomorphin, prodynorphins, transglutaminase, and gliadin-bound transglutaminase (gliadin-transglutaminase complex).
  • assays and assay kits of particular interest allow for testing IgA and/or IgG antibodies to one or more wheat antigens, a ⁇ -gliadin protein or one or more peptides thereof such as ⁇ -gliadin-33-mer and/or ⁇ -gliadin-17-mer, a ⁇ -gliadin protein or one or more peptides thereof such as ⁇ -gliadin-15-mer, an ⁇ -gliadin protein or one or more peptides thereof such as ⁇ -gliadin-17-mer, wheat germ agglutinin, an opioid peptide such as one or more of gluteomorphin, prodynorphin and/or dynorphin, a glutenin protein or one or more peptides thereof such as glutenin-21-mer, a de-amidated glutenin protein or one or more peptides thereof, a gliadin-transglutaminase complex, or combinations thereof.
  • 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.
  • 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.
  • FIG. 1 is a schematic showing differentiation between celiac disease (right hand listing) and gluten immune reactivity/sensitivity (left hand listing), as contemplated herein.
  • FIG. 2 is a diagram showing the layout of a sample microtiter plate having 12 different rows with 12 different antigens and peptides.
  • FIG. 3 is a diagram showing the layout of a sample microtiter plate by which IgG or IgA is measured against 12 different antigens or peptides from wheat and associated tissue antigens (antigens or peptides are transparent).
  • FIG. 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 purposes (antigens or peptides are transparent).
  • FIG. 5 is a diagram of an antibody testing protocol for celiac disease using tTg and various antigens according to the prior art (17).
  • FIG. 6 is a diagram of a testing protocol for celiac disease using deamidated gliadin peptide according to the prior art (17).
  • FIG. 7 is a diagram of a proposed testing protocol for celiac disease, gluten immune reactivity/sensitivity and autoimmunity using a repertoire of wheat antigens and peptides 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 pathologies as opposed to the classical celiac disease against an array of wheat antigens and peptides.
  • 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 ⁇ -gliadin protein or one or more peptides thereof; a ⁇ -gliadin protein or one or more peptides thereof; an ⁇ -gliadin protein or one or more peptides thereof; a glutenin protein or one or more peptides thereof; one or more opioid peptides; a gliadin-transglutaminase complex (gliadin bound to transglutaminase); transglutaminase; wheat germ agglutinin; and combinations thereof.
  • IgG immunoglobulin G
  • IgA immunoglobulin A
  • the whole-wheat antigen is deamidated and can be prepared by combining water-soluble and alcohol-soluble whole-wheat proteins.
  • the ⁇ -gliadin protein or one or more peptides thereof includes ⁇ -gliadin-33-mer and/or ⁇ -gliadin-17-mer, with other ⁇ -gliadin peptides contemplated.
  • the ⁇ -gliadin protein or one or more peptides thereof includes ⁇ -gliadin-15-mer, with other ⁇ -gliadin peptides contemplated.
  • the ⁇ -gliadin protein or one or more peptides thereof includes ⁇ -gliadin-17-mer, with other ⁇ -gliadin peptides contemplated.
  • the glutenin protein or one or more peptides thereof includes glutenin-21-mer, with other glutenin peptides contemplated.
  • the one or more opioid peptides includes exorphin peptides including gluteomorphin, prodynorphin and/or dynorphin, with other exorphin peptides contemplated.
  • 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 antigen; 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 particularly interesting where the wheat antigen is deamidated, and the gliadin antigen is selected from the group consisting of an ⁇ -gliadin-33-mer, an ⁇ -gliadin-17-mer, a ⁇ -gliadin-15-mer, an ⁇ -gliadin-17-mer, and a glutenin-21-mer.
  • Test plates and kits can advantageously test for antigens to at least three, five, seven or all of ⁇ -gliadin, ⁇ -gliadin, ⁇ -gliadin, glutenin, wheat germ agglutinin, gluteomorphin, prodynorphins, transglutaminase, and gliadin-bound transglutaminase.
  • assays and assay kits of particular interest allow for testing IgA and/or IgG antibodies to one or more wheat antigens, a ⁇ -gliadin protein or one or more peptides thereof such as ⁇ -gliadin-33-mer and/or ⁇ -gliadin-17-mer, a ⁇ -gliadin protein or one or more peptides thereof such as ⁇ -gliadin-15-mer, an ⁇ -gliadin protein or one or more peptides thereof such as ⁇ -gliadin-17-mer, wheat germ agglutinin, an opioid peptide such as one or more of gluteomorphin, prodynorphin and/or dynorphin, a glutenin protein or one or more peptides thereof such as glutenin-21-mer, a de-amidated glutenin protein or one or more peptides thereof, a gliadin-transglutaminase complex, or combinations thereof.
  • 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.
  • 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.
  • Wheat antigens and peptides A whole-wheat antigen was prepared by combining water-soluble and alcohol-soluble proteins. Different gliadin peptides including ⁇ -gliadin 33-mer, 17-mer, ⁇ -gliadin 15-mer, ⁇ -gliadin 17-mer, gluteomorphin, prodynorphin, transglutaminases, and gliadin bound to transglutaminase, glutamic acid decarboxylase (GAD-65) HPLC grade were synthesized by Bio-Synthesis Inc. (Lewisville, Tex.). Wheat germ agglutinin (WGA) was purchased 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 ⁇ l were added to each well of a polystyrene flat-bottom ELISA plate.
  • the ELISA plates were incubated overnight at 4° C. and then washed three times with 200 ⁇ l Tris-buffered saline (TBS) containing 0.05% Tween 20 (pH 7.4).
  • TBS Tris-buffered saline
  • the non-specific binding of immunoglobulins was prevented by adding 200 mL of 2% bovine serum albumin (BSA) in TBS, and incubated overnight at 4° C. Plates were washed and after conducting quality control were kept at 4° C. until used.
  • BSA bovine serum albumin
  • the enzyme conjugates included: Affinity Purified Antibody Phosphatase-labeled Goat anti-Human IgG (Jackson ImmunoResearch, Cat#109-055-008), and Affinity Purified Antibody Phosphatase-labeled Goat anti-Human IgA (Jackson ImmunoResearch, Cat#109-055-011).
  • Additional reagents and materials included in the method as further described herein includes: Phosphate-Buffered Saline Powder (Sigma, Cat#P3813-10PAK), Bovine Serum Albumin (Biocell, Cat#3203-00), Sodium Azide (Sigma, Cat#S-2002), Tween 20 (Sigma, Cat#P1379-1000 ML), Glycerol (Sigma, Cat#G5516-500 ML), Sodium Hydroxide (Sigma, Cat#S-5881), Magnesium Chloride (Sigma, Cat#8266), Diethanolamine (Sigma, Cat#D-8885), 1.0 N Hydrochloric Acid Solution (Sigma, Cat#H3162), 5 mg Substrate Tablets: p-NPP (para-nitophenyl phosphate) (Sigma, Cat#S-0942), and Distilled water (D. H 2 O).
  • Phosphate-Buffered Saline Powder Sigma, Cat#P3813-10PAK
  • 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-associated 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/IgA unbound conjugate were removed by a further 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.
  • PNPP paranitrophenyl phosphate
  • Sodium hydroxide was added to each well to stop the reaction. The intensity of color was read at 405 nm.
  • Plain red tops or red tiger tops were used for specimen collection, although in certain aspects, other specimen collection apparatus are contemplated for this assay.
  • Blood samples were collected using aseptic venipuncture techniques and serum was obtained using standard procedures. In certain aspects, it is preferred that a minimum of about 50 ⁇ l of serum for the assay, which therefore corresponds to about 0.5 ml or more of blood.
  • the test assay procedure includes preparing the desired number of coated wells or plates with the desired number and type of wheat-associated antigens and/or peptides. Once the microtiter wells are prepared, about 100 ⁇ l of 1:100 diluted control calibrator are added to Rows A and B of the microtiter plate as shown in FIG. 3 using a multi-channel pipettor. About 100 ⁇ l of 1:100 diluted patient's test sample, here blood serum, was added to duplicate wells of rows C and D for Clinical Specimen 1, rows E and F for Clinical Specimen 2, and rows G and H for Clinical Specimen 3.
  • the plates were then incubated for 60 minutes at room temperature. After incubation, the wells were emptied and washed four times with PBS using an ELISA Washer. About 100 ⁇ l of optimally diluted alkaline phosphatase-labeled goat anti-human IgA was added to the IgA plate or about 100 ⁇ l of enzyme-labeled IgG was added to the IgG plate at optimal dilution.
  • a substrate solution was prepared by mixing 5 mg of a p-nitrophenyl phosphate tablet with 5 ml 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 ⁇ l of substrate solution was added to each well. The plate was then incubated for 30 minutes at room temperature with the avoidance of any exposure to direct sunlight. The reaction was stopped by adding about 50 ⁇ l of 3 N NaOH. The color intensity of the wells were read using a microtiter plate reader at 405 nm against a blank well, with the absorbance values of the calibrators, controls and unknown samples being recorded.
  • 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 C1 and D1 by the mean OD of wells A1 and B1, the mean OD of wells C2 and D2 by the mean OD of wells A2 and B2, the mean OD of wells C3 and D3 by the mean OD of wells A3 and B3, etc.).
  • IgG and IgA antibody patterns of 3 patients with celiac disease, 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.
  • celiac disease can be differentiated from silent celiac disease, gluten immune reactivity/sensitivity and/or gluten-related autoimmunity as follows:
  • test results include positive results of IgA and/or IgG against any or combination of wheat proteins, ⁇ -gliadin 33-mer, deamidated ⁇ -gliadin 33-mer, ⁇ -gliadin 25-mer, ⁇ -gliadin 18-mer, ⁇ -gliadin 17-mer, ⁇ -gliadin 15-mer, ⁇ -gliadin 17-mer, glutenin, deamidated glutenin, gluteomorphin, prodynorphin and wheat germ agglutinin, and negative results against tranglutaminase-2, and at least one of IgA and IgG tests positive against at least one of transglutaminase-3 and transglutaminase-6.
  • test results include positive results for IgG and/or IgA against wheat antigens, in particular native ⁇ -gliadin, ⁇ -gliadin, ⁇ -gliadin, glutenin, deamidated glutenin, gluteomorphin, and wheat germ agglutinin, but not deamidated ⁇ -gliadin and not transglutaminase-2.
  • test results include a positive for IgG and/or IgA against wheat antigens, in particular native ⁇ -gliadin, ⁇ -gliadin, ⁇ -gliadin, glutenin, deamidated glutenin, gluteomorphin, and wheat germ agglutinin, and positive results for any IgG, IgA or IgM to glutamic acid decarboxylase, cerebellar tissue antigens or peptides or other tissue antigens, and negative results for IgA to deamidated ⁇ -gliadin, deamidated glutenin or against transglutaminase-2, 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 standards above the mean, while IgA antibody against 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 measured against ⁇ -gliadin 33-mer the IgG antibody level was significantly elevated in one and the IgA level in two out of three patients (Tables 2, 3).
  • the IgA immune reaction against wheat antigens and peptides in combination with tTg and gliadin-tTg complex clearly distinguished between celiac disease and gluten immune reactivity/sensitivity, in which IgG and/or IgA antibodies are 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 patients with Crohn's disease.
  • the pattern of IgG and IgA antibodies against tTg and gliadin-tTg complex clearly shows that these patients, in addition to Crohn's disease, are also suffering from gluten immune reactivity/sensitivity, and possibly also celiac disease (Tables 6, 7).
  • celiac disease 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 disease is perceived as presenting GI symptoms accompanied by malabsorption. But many patients with celiac disease do not present GI symptoms. These individuals may have silent or atypical celiac disease, and the condition may present with iron deficiency, anemia, increased liver enzymes, osteoporosis or neurological symptoms (12). As used herein, the term “atypical celiac disease” refers to celiac disease in patients who have only subtle symptoms, and the term “silent celiac disease” refers to celiac disease in patients who are asymptomatic.
  • 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 anti-tTg 2.1 U/mL normal range ⁇ 6 U/mL
  • IgA anti-tTg 1.2 U/mL normal range ⁇ 4 U/mL
  • negative for HLA DQ2 and DQ8 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 anti-tTg 2.1 U/mL normal range ⁇ 6 U/mL
  • IgA anti-tTg 1.2 U/mL normal range ⁇ 4 U/mL
  • negative for HLA DQ2 and DQ8 negative for HLA DQ2 and DQ8.
  • gluten sensitivity refers to a state of heightened immunological responsiveness to gluten as indicated by the elevation of IgG, IgA or both against gliadin but not against transglutaminase (14).
  • Gluten sensitivity begins with the loss of mucosal immune tolerance to wheat antigens and peptides due to environmental factors affecting the mucosal immune homeostasis.
  • gluten sensitivity was confirmed based on GI symptoms and immunological testing, in particular IgG and IgA against gliadin and its associated proteins and peptides almost 100 days after the triggering factors 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 dysregulation of her mucosal immune system, followed by a breakdown in immunological tolerance to wheat and other dietary proteins and peptides.
  • her hemoglobin level continued to be low, while her ESR fluctuated.
  • Close questioning 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 ⁇ g/L (normal range 10-150 ⁇ g/L), which confirmed iron deficiency.
  • MRI scan of the brain showed extensive white matter abnormalities not typical of multiple sclerosis, but no abnormalities were detected in CSF examination.
  • This comprehensive gluten sensitivity and immune reactivity screen revealed IgG against wheat antigens, ⁇ -gliadin 33- and 17-mer, ⁇ - and ⁇ -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 IgA and IgG tested against tTg were within the normal range.
  • 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).
  • the IgG antibody elevations were considered non-specific or protective, and the patient was put on painkillers and sent home with no diagnosis of any specific disorder.
  • FIGS. 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 al. (17).

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