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

Abstract

Antibodies are used as biomarkers to assist in distinguishing gluten immune reactivity and sensitivity, silent celiac disease, Crohn's disease and other gut-related pathologies from classical celiac disease. In one class of embodiments, 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 deamidated glutenin, a prodynorphin, and a dynorphin. Test results are considered particularly interesting where the wheat antigen and the gliadin antigen are both selected from the group consisting of an α-gliadin-33-mer, an α-gliadin-17-mer, a γ-gliadin-15-mer, an ω-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 α-gliadin, γ-gliadin, ω-gliadin, glutenin, wheat germ agglutinin, gluteomorphin, prodynorphins, transglutaminase-2, transglutaminase-3, transglutaminase-6, and gliadin-bound transglutaminase.

1 . A method of testing a sample from an animal, comprising:
 measuring a first signal derived from binding of a first fraction of the sample to a wheat antigen;   measuring a second signal derived from binding of a second fraction of the sample to a gliadin antigen; and   measuring a third signal derived from binding of a third fraction 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.   
     
     
         2 . The method of  claim 1 , wherein the sample comprises blood serum. 
     
     
         3 . The method of  claim 1 , wherein the sample comprises saliva. 
     
     
         4 . The method of  claim 1 , wherein the wheat antigen is selected from the group consisting of an α-gliadin-33-mer, an α-gliadin-17-mer, a γ-gliadin-15-mer, an ω-gliadin-17-mer, and a glutenin-21-mer. 
     
     
         5 . The method of  claim 4 , wherein at least one of the wheat antigens is deamidated. 
     
     
         6 . The method of  claim 1 , wherein the animal is a human being. 
     
     
         7 . A method of making a determination that assists in differentiating from celiac disease from (a) gluten immune reactivity and sensitivity and (b) a diagnosis related to gluten immune reactivity/sensitivity and autoimmunity, using test results derived from analyzing a sample obtained from a human, comprising:
 testing or having the sample tested for IgA against deamidated α-gliadin 33-mer or transglutaminase-2; and   testing or having the sample tested for IgA against deamidated glutenin 21-mer.   
     
     
         8 . The method of  claim 7  further comprising testing or having the sample tested for a serological pattern indicative of silent celiac disease where:
 at least one of IgA and IgG test positive against at least one of α-gliadin 33-mer, deamidated α-gliadin 33-mer, α-gliadin 25-mer, α-gliadin 18-mer, α-gliadin 17-mer, γ-gliadin 15-mer, ω-gliadin 17-mer, glutenin, deamidated glutenin, gluteomorphin, prodynorphin and wheat germ agglutinin; and 
 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. 
 
     
     
         9 . The method of  claim 7  further comprising testing or having the sample tested for a serological pattern indicative of gluten immune reactivity and sensitivity where:
 at least one of IgA and IgG test positive against at least one of α-gliadin, γ-gliadin, ω-gliadin, glutenin, deamidated glutenin, gluteomorphin, and wheat germ agglutinin; and 
 both IgA and IgG test negative against deamidated α-gliadin, deamidated glutenin or transglutaminase-2. 
 
     
     
         10 . The method of  claim 7  further comprising testing or having the sample tested for a serological pattern indicative of a diagnosis related to gluten immune reactivity/sensitivity and autoimmunity where:
 at least one of IgA and IgG test positive against at least one of native α-gliadin, γ-gliadin, 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; and 
 IgA tests negative against deamidated α-gliadin, deamidated glutenin or transglutaminase. 
 
     
     
         11 . A method of screening for the presence of at least one of IgG and IgA antibodies in a patient, comprising:
 obtaining test results derived from measuring first, second and third signals, where the first signal is derived from binding of a first fraction of the sample to a wheat antigen, the second signal is derived from binding of a second fraction of the sample to a gliadin antigen; and the third signal is derived from binding of a third fraction of the sample to an antigen selected from the group consisting of: (1) a wheat germ agglutinin; (2) gluteomorphin; (3) a glutenin; (4) a de-aminated glutinin; (5) a prodynorphin; and (6) a dynorphin; and   using the test results of  claim 1  to ascertain whether the patient has the antibodies.   
     
     
         12 . The method of  claim 11 , wherein the antibodies bind to wheat and gliadin peptides. 
     
     
         13 . The method of  claim 11 , wherein the antibodies bind to alcohol-soluble and/or water-soluble components of wheat, and the gliadin antigen is selected from the group consisting of α-gliadin-33-mer, α-gliadin-17-mer, γ-gliadin-15-mer, ω-gliadin-17-mer, and a glutenin-21-mer. 
     
     
         14 . The method of  claim 11 , further comprising using the test results to assist in ascertaining whether the patient has celiac disease. 
     
     
         15 . The method of  claim 14 , further comprising securing results from testing the sample for at least five of antibodies to α-gliadin, γ-gliadin, ω-gliadin, glutenin, wheat germ agglutinin, gluteomorphin, prodynorphins, transglutaminase, and gliadin-bound transglutaminase. 
     
     
         16 . The method of  claim 11 , further comprising using the test results to assist in distinguishing whether the patient has silent or atypical celiac disease, as opposed to patently symptomatic celiac disease. 
     
     
         17 . The method of  claim 16 , further comprising securing results from testing the sample for at least five of antibodies to α-gliadin, γ-gliadin, ω-gliadin, glutenin, wheat germ agglutinin, gluteomorphin, prodynorphins, transglutaminase, and gliadin-bound transglutaminase. 
     
     
         18 . The method of  claim 11 , further comprising using the test results to assist in ascertaining whether the patient has at least one of gluten immune reactivity and sensitivity and autoimmunity. 
     
     
         19 . The method of  claim 18 , further comprising securing results from testing the sample for at least five of antibodies to α-gliadin, γ-gliadin, ω-gliadin, glutenin, wheat germ agglutinin, gluteomorphin, prodynorphins, transglutaminase, and gliadin-bound transglutaminase. 
     
     
         20 . The method of  claim 11 , further comprising using the test results to assist in ascertaining whether the patient has at least one of Crohn's disease and chronic immune activation. 
     
     
         21 . The method of  claim 20 , further comprising securing results from testing the sample for at least five of antibodies to α-gliadin, γ-gliadin, ω-gliadin, glutenin, wheat germ agglutinin, gluteomorphin, prodynorphins, transglutaminase, and gliadin-bound transglutaminase. 
     
     
         22 . A method of screening for the presence of IgG and/or IgA antibodies in a patient, comprising providing or obtaining test results derived from measuring signals derived from binding of a patient's antibodies to (a) at least one of α-gliadin and transglutaminase, and (b) at least one of water- and alcohol-soluble components of wheat, wheat germ agglutinin, ω- and γ-gliadins, glutenin, gluteomorphin, and gliadin-bound transglutaminase; and using the test results of  claim 1  to ascertain whether the patient has the antibodies. 
     
     
         23 . A test plate containing at least three bound test substances selected from the list consisting of water- and alcohol-soluble components of wheat, α-gliadin-33-mer, α-gliadin-17-mer, γ-gliadin-15-mer, ω-gliadin-17-mer, glutenin-21-mer, gluteomorphin 16-mer, prodynorphin, gliadin-bound transglutaminase, and wheat germ agglutinin. 
     
     
         24 . The test plate of  claim 23 , containing at least five of the bound test substances. 
     
     
         25 . The test plate of  claim 23 , containing at least seven of the bound test substances. 
     
     
         26 . The test plate of  claim 23 , containing each of the bound test substances. 
     
     
         27 . A test kit comprising the test plate of  claim 23 , and instructions for using the test plate to measure IgG and/or IgA antibodies.