U.S. patent application number 13/354119 was filed with the patent office on 2012-07-26 for methods and apparatus for detection of gluten sensitivity, and its differentiation from celiac disease.
Invention is credited to Aristo Vojdani.
Application Number | 20120190571 13/354119 |
Document ID | / |
Family ID | 46516373 |
Filed Date | 2012-07-26 |
United States Patent
Application |
20120190571 |
Kind Code |
A1 |
Vojdani; Aristo |
July 26, 2012 |
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
.alpha.-gliadin-33-mer, an .alpha.-gliadin-17-mer, a
.gamma.-gliadin-15-mer, an .omega.-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 .alpha.-gliadin,
.gamma.-gliadin, .omega.-gliadin, glutenin, wheat germ agglutinin,
gluteomorphin, prodynorphins, transglutaminase-2,
transglutaminase-3, transglutaminase-6, and gliadin-bound
transglutaminase.
Inventors: |
Vojdani; Aristo; (Los
Angeles, CA) |
Family ID: |
46516373 |
Appl. No.: |
13/354119 |
Filed: |
January 19, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61434501 |
Jan 20, 2011 |
|
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|
Current U.S.
Class: |
506/9 ;
506/18 |
Current CPC
Class: |
G01N 33/6854 20130101;
G01N 2800/065 20130101; G01N 2800/24 20130101 |
Class at
Publication: |
506/9 ;
506/18 |
International
Class: |
C40B 30/04 20060101
C40B030/04; C40B 40/10 20060101 C40B040/10 |
Claims
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 .alpha.-gliadin-33-mer, an
.alpha.-gliadin-17-mer, a .gamma.-gliadin-15-mer, an
.omega.-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
.alpha.-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 .alpha.-gliadin 33-mer, deamidated .alpha.-gliadin
33-mer, .alpha.-gliadin 25-mer, .alpha.-gliadin 18-mer,
.alpha.-gliadin 17-mer, .gamma.-gliadin 15-mer, .omega.-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 .alpha.-gliadin, .gamma.-gliadin,
.omega.-gliadin, glutenin, deamidated glutenin, gluteomorphin, and
wheat germ agglutinin; and both IgA and IgG test negative against
deamidated .alpha.-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 .alpha.-gliadin, .gamma.-gliadin, .omega.-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 .alpha.-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
.alpha.-gliadin-33-mer, .alpha.-gliadin-17-mer,
.gamma.-gliadin-15-mer, .omega.-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
.alpha.-gliadin, .gamma.-gliadin, .omega.-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
.alpha.-gliadin, .gamma.-gliadin, .omega.-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
.alpha.-gliadin, .gamma.-gliadin, .omega.-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
.alpha.-gliadin, .gamma.-gliadin, .omega.-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 .alpha.-gliadin and
transglutaminase, and (b) at least one of water- and
alcohol-soluble components of wheat, wheat germ agglutinin,
.omega.- and .gamma.-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, .alpha.-gliadin-33-mer,
.alpha.-gliadin-17-mer, .gamma.-gliadin-15-mer,
.omega.-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.
Description
RELATED APPLICATION
[0001] The present application claims the benefit of U.S.
Provisional Application No. 61/434,501 filed Jan. 20, 2011, which
is incorporated herein in its entirety by reference.
FIELD OF THE INVENTION
[0002] 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.
BACKGROUND OF THE INVENTION
[0003] 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 reaction to gluten
is mediated by both cellular and humoral immune responses,
resulting in the presentation of different symptomatologies. For
example, in wheat allergy a specific sequence of gliadin peptides
cross-links two IgE molecules on the surface of mast cells and
basophils that trigger the release of mediators such as histamines
and leukotrienes (3).
[0004] 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.
[0005] 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.
Throughout this application, unless the context dictates the
contrary, all ranges set forth herein should be interpreted as
being inclusive of their endpoints, and open-ended ranges should be
interpreted to include commercially practical values. Similarly,
all lists of values should be considered as inclusive of
intermediate values unless the context indicates the contrary.
[0006] Markers for confirming a diagnosis of this disorder are IgA
against native, deamidated gliadin peptides and IgA anti-tissue
transglutaminase (tTg) autoantibody. In 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 objective 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
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-.gamma., 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) and
T-cell-mediated reaction, which is called celiac disease or gluten
sensitivity with enteropathy (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 development of gluten sensitivity (7). Due
to antigenic similarities between 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 (8-10).
[0007] It should be appreciated that the term "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.
[0008] While GS patients, similar to CD patients, are unable to
tolerate 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 picture 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 clinicians makes GS an
extremely dangerous disorder. This is because the persistence of
IgG and/or IgA antibodies in the blood for long periods of time,
along with inducers of inflammatory cascades can result in
full-blown autoimmunity. If this were to be the case, 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.
[0009] A comparison between celiac disease and gluten immune
reactivity/sensitivity is shown in FIG. 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.sup.+), 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 gliadin results in
the 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.sup.- and HLA
DQ2/DQ8.sup.+.
[0010] 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 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.
[0011] 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 proteins and
peptides may activate an inflammatory cascade. In the absence of
tTg activation, however, villous atrophy does not occur.
Furthermore, gliadin peptides do not go through de-amidation, and
consequently IgG and IgA antibodies are produced only against
native wheat and gliadin peptides.
[0012] With continuous exposure to wheat antigens and continuous
mucosal immune tolerance, 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.
[0013] Furthermore, 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.
[0014] Thus, a new paradigm is needed for aid in diagnosing and
distinguishing among various gut-related diseases, including gluten
immune reactivity and sensitivity, silent celiac disease, celiac
disease, and gut-related autoimmunity.
SUMMARY OF THE INVENTION
[0015] 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.
[0016] In certain aspects of the present invention, 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.
[0017] 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
gliadin antigen is selected from the group consisting of an
.alpha.-gliadin-33-mer, an .alpha.-gliadin-17-mer, a
.gamma.-gliadin-15-mer, an .omega.-gliadin-17-mer, and a
glutenin-21-mer.
[0018] In certain aspects of the present 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 .gamma.-gliadin
protein or a peptide thereof such as .gamma.-gliadin-15-mer, an
.omega.-gliadin protein or a peptide thereof such as
.omega.-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.
[0019] In certain aspects of the present invention, 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
.alpha.-gliadin, .gamma.-gliadin, .omega.-gliadin, glutenin, wheat
germ agglutinin, gluteomorphin, prodynorphins, transglutaminase,
and gliadin-bound transglutaminase (gliadin-transglutaminase
complex).
[0020] 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 .alpha.-gliadin protein
or one or more peptides thereof such as .alpha.-gliadin-33-mer
and/or .alpha.-gliadin-17-mer, a .gamma.-gliadin protein or one or
more peptides thereof such as .gamma.-gliadin-15-mer, an
.omega.-gliadin protein or one or more peptides thereof such as
.omega.-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.
[0021] 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.
[0022] Various objects, features, aspects and advantages of the
inventive subject matter of the present invention will become more
apparent from the following detailed description of preferred
embodiments, along with the accompanying drawing figures in which
like numerals represent like components.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIG. 1 is a schematic showing differentiation between celiac
disease (right hand listing) and gluten immune
reactivity/sensitivity (left hand listing), as contemplated
herein.
[0024] FIG. 2 is a diagram showing the layout of a sample
microtiter plate having 12 different rows with 12 different
antigens and peptides.
[0025] 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).
[0026] 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).
[0027] FIG. 5 is a diagram of an antibody testing protocol for
celiac disease using tTg and various antigens according to the
prior art (17).
[0028] FIG. 6 is a diagram of a testing protocol for celiac disease
using deamidated gliadin peptide according to the prior art
(17).
[0029] 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.
DETAILED DESCRIPTION OF THE DRAWINGS
[0030] 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.
[0031] 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
.alpha.-gliadin protein or one or more peptides thereof; a
.gamma.-gliadin protein or one or more peptides thereof; an
.omega.-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.
[0032] In certain aspects of the present invention, the whole-wheat
antigen is deamidated and can be prepared by combining
water-soluble and alcohol-soluble whole-wheat proteins. The
.alpha.-gliadin protein or one or more peptides thereof includes
.alpha.-gliadin-33-mer and/or .alpha.-gliadin-17-mer, with other
.alpha.-gliadin peptides contemplated. The .gamma.-gliadin protein
or one or more peptides thereof includes .gamma.-gliadin-15-mer,
with other .gamma.-gliadin peptides contemplated. The
.omega.-gliadin protein or one or more peptides thereof includes
.omega.-gliadin-17-mer, with other .omega.-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.
[0033] In certain aspects of the present invention, 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
.alpha.-gliadin-33-mer, an .alpha.-gliadin-17-mer, a
.gamma.-gliadin-15-mer, an .omega.-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 .alpha.-gliadin,
.gamma.-gliadin, .omega.-gliadin, glutenin, wheat germ agglutinin,
gluteomorphin, prodynorphins, transglutaminase, and gliadin-bound
transglutaminase.
[0034] 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 .alpha.-gliadin protein
or one or more peptides thereof such as .alpha.-gliadin-33-mer
and/or .alpha.-gliadin-17-mer, a .gamma.-gliadin protein or one or
more peptides thereof such as .gamma.-gliadin-15-mer, an
.omega.-gliadin protein or one or more peptides thereof such as
.omega.-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.
[0035] 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.
[0036] 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, the preferred method and materials are now described in
the exemplary description of assays to further illustrate the
present invention.
Example 1
ELISA Assay
[0037] A. Materials And Methods--Plate and Sample Preparation:
[0038] Wheat antigens and peptides. A whole-wheat antigen was
prepared by combining water-soluble and alcohol-soluble proteins.
Different gliadin peptides including .alpha.-gliadin 33-mer,
17-mer, .gamma.-gliadin 15-mer, .omega.-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.).
[0039] 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 .mu.l were added to
each well of a polystyrene flat-bottom ELISA plate.
[0040] The ELISA plates were incubated overnight at 4.degree. C.
and then washed three times with 200 .mu.l 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
serum albumin (BSA) in TBS, and incubated overnight at 4.degree. C.
Plates were washed and after conducting quality control were kept
at 4.degree. C. until used.
[0041] 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).
[0042] Other 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.sub.2O).
[0043] 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-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.
[0044] 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 reaction. The intensity of color was read at 405
nm.
[0045] Plain red tops or red tiger tops (SST tubes) were used for
specimen collection, although in certain aspects, other specimen
collection apparatus are contemplated for this assay.
[0046] 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 .mu.l
of serum for the assay, which therefore corresponds to about 0.5 ml
or more of blood.
[0047] B. Test Assay Procedure
[0048] The analytical procedure for IgG and/or IgA antibody array
to assist in diagnosing and detection of gluten immune reactivity
and sensitivity, silent celiac disease, Crohn's disease and other
gut-related pathologies is now discussed. In some aspects, all
reagents were allowed to reach room temperature before the test
assay was commenced. 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 .mu.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
.mu.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.
[0049] On a separate plate, the periodic negative and positive
controls similar to clinical specimens in duplicates were
conducted, as shown in FIG. 4.
[0050] 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 .mu.l of
optimally diluted alkaline phosphatase-labeled goat anti-human IgA
was added to the IgA plate or about 100 .mu.l of enzyme-labeled IgG
was added to the IgG plate at optimal dilution.
[0051] The respective plates was then incubated for 30-60 minutes
at room temperature. About ten minutes before the
conjugate-incubation ends, 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 .mu.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 .mu.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.
[0052] C. Calculation of Results
[0053] After the plate was read the plate at 405 nm to obtain the
optical density values (OD405), the mean ODs of the negative
controls, the mean ODs 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).
[0054] 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.).
[0055] The results were then compared to the established reference
ranges.
TABLE-US-00001 Index = Mean OD of patients Mean OD of calibrators
##EQU00001## Index Calculation For Wheat Antitens Cal 1 (OD) 0.41
Cal 2 OD 0.44 Sample 3 A (OD) 3.77 Sample 3 B OD 3.79 Index
8.93
[0056] D. Interpretation of Results
[0057] Examples of 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.
[0058] 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:
[0059] a. A serological pattern of silent celiac disease is
indicated where the test results include positive results of IgA
and/or IgG against any or combination of wheat proteins,
.alpha.-gliadin 33-mer, deamidated .alpha.-gliadin 33-mer,
.alpha.-gliadin 25-mer, .alpha.-gliadin 18-mer, .alpha.-gliadin
17-mer, .gamma.-gliadin 15-mer, .omega.-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.
[0060] b. A serological pattern of gluten immune reactivity and
sensitivity is indicated where the test results include positive
results for IgG and/or IgA against wheat antigens, in particular
native .alpha.-gliadin, .gamma.-gliadin, .omega.-gliadin, glutenin,
deamidated glutenin, gluteomorphin, and wheat germ agglutinin, but
not deamidated .alpha.-gliadin and not transglutaminase-2.
[0061] c. A diagnosis related to gluten immune
reactivity/sensitivity and autoimmunity is indicated where the test
results include a positive for IgG and/or IgA against wheat
antigens, in particular native .alpha.-gliadin, .gamma.-gliadin,
.omega.-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 .alpha.-gliadin, deamidated glutenin or against
transglutaminase-2, but positive IgA or IgG against
transglutaminase-3 and/or transglutaminase-6.
[0062] 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
.alpha.-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).
[0063] When compared to patients with celiac disease, in patients
with gluten immune reactivity and sensitivity none of the three
patients showed a significant IgA reactivity against tTg and
gliadin-tTg complex, while the IgG and IgA antibodies against wheat
antigens and in combination with one or more wheat peptides
(.alpha.-gliadin, .gamma.-gliadin, .omega.-gliadin, glutenin,
gluteomorphin, prodynorphin and wheat germ agglutinin) was
significantly elevated. 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).
[0064] 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).
CASE STUDY EXAMPLES
[0065] 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.
[0066] 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
[0067] A 76 year-old man with longstanding dyspepsia, indigestion,
tiredness and rapid weight loss was referred for gastrointestinal
evaluation. Blood tests showed macrocytic anemia with low
concentrations of folate and vitamin B-12. The patient's hemoglobin
concentration was 79 g/L, albumin 32 g/L, and transglutaminase 212
.mu.g/mL (normal range=0-10 .mu.g/mL. An urgent colonoscopy and
duodenal 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 .alpha.-gliadin were very high; against
transglutaminase, IgA but not IgG was 3.8-fold higher than the
reference range. In view of the IgA positivity against gliadin and
transglutaminase and diagnosis of celiac disease he was transfused
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 transglutaminase the repeat tests for these antibodies
were negative, which is a further indication that disease
management plus a gluten-free diet was instrumental in the
treatment of this elderly patient with a silent celiac disease.
[0068] Discussion: According to Catassi et al. (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 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.
[0069] The increasing recognition of celiac disease is attributed
to the use of new serological assays with higher sensitivity and
specificity. Until recently celiac disease was incorrectly
perceived as being uncommon and detected mainly during infancy or
childhood. However, it is now recognized that most cases of CD
occur in adults 40-60 years old. Patients in this age group may
present their symptoms, lab test results and other 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).
[0070] Consequently, as this case shows, if an adult patient
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 improvement in symptoms, the patient should
commit to the diet regardless of age.
[0071] B. Case Report #2: Gluten Immune Reactivity and Sensitivity
Induced by a Combination of Anesthetics, Antibiotics and Pain
Medication
[0072] A 46 year-old woman was given her yearly checkup by her
internist. Based on her 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 different visits over ten days she was
treated with anesthetic material (mepivacaine), antibiotics 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 the lips and periorbital area swelling. The
patient became agitated and exhibited with a 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 the allergic reaction was controlled, the patient developed
severe vomiting and diarrhea with severe abdominal pain, which
lasted for 8 days. Two weeks later, while the diarrhea had
ameliorated, the patient continued to complain about bloating and
abdominal discomfort with irritable bowel-like syndrome. She was
referred to a GI specialist who detected nothing of note upon a
thorough examination. The possibility of gluten sensitivity was
then considered, and the patient was tested for HLA typing and IgG
and IgA anti-gliadin and anti-transglutaminase antibodies.
Immunological tests showed these results: 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); and negative for HLA
DQ2 and DQ8. Based on these findings gluten sensitivity and celiac
disease 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 her 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 gliadin were 4 to 9-fold
above the reference range (gliadin IgG=79 U/mL; IgA=54 U/mL).
However, IgG and IgA antibodies against tTg were within the normal
range. In addition, the IgG and IgA antibody testing was performed
against an array of wheat, gliadin, glutenin, wheat germ
agglutinin, gliadin-tTg complex and tTg antigens. 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 tTg and 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 against gliadin but
not against transglutaminase showed that the patient, due to
allergic reaction to environmental factors, had lost tolerance to
wheat antigens and had developed gluten sensitivity but not celiac
disease. Despite the absence of elevation in IgG and IgA levels
against tTg, due to the continuous GI discomfort and the elevated
IgG and IgA against gliadin, a gluten-free diet was recommended,
and a dietitian advised the patient to take probiotics and go on a
restricted diet free of glutens and also of lectins, since the WGA
level was also elevated for both IgG and IgA. Six months after the
introduction of the diet and the probiotics, the patient's GI
discomfort had subsided and she was back to normal health.
[0073] Discussion: The term 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.
[0074] In this case 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. This,
possibly 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
process gliadin-specific B cells are assisted by gliadin-specific T
cells, leading to B-cell clonal expansion and the release of IgG
and IgA antibodies to gliadin and associated proteins and peptides,
which in this case was detected about 100 days after the original
traumatic experience.
[0075] It is concluded herein that screening for gluten sensitivity
in patients with GI discomfort associated with the use of
anesthetics 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
deprive the patient of an accurate diagnosis and the proper
treatment by implementation of a gluten-free diet, but may also
result in unnecessary medical interventions with their associated
side effects.
[0076] C. Case Report #3: Gluten Immune Reactivity, Sensitivity and
Autoimmunity
[0077] Here, a case report is described in which the original
presentation led to an erroneous diagnosis of irritable bowel
syndrome, resulting in incorrect medical intervention. The correct
diagnosis of gluten immune reactivity and sensitivity was made
after years of mistreatment.
[0078] 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 10.8 g/dl and normal
chemistry profile including liver enzyme. Over several visits
detailed biochemical and immunological profiles including 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 .beta.-blockers
and nexium, which moderately improved her symptomatologies. Four
years later, however, in addition to the old GI symptoms and
headache, she presented symptoms of malaise, blurred 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/1st hour), ANA of 1:80 (normal range
<40), mild elevation in IgA smooth muscle antibody,
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 difficulty
in passing urine accompanied by tingling and sensory disturbance in
her trunk and legs, which led to her being referred to a
neurologist. 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 .mu.g/L (normal range 10-150 .mu.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. While blood and
CSF examination showed no evidence of bacterial and viral infection
including syphilis, mycobacteria, borrelia, EBV, CMV, HTLV, and
Herpes Type-6, visual evoked potentials showed delay in both optic
nerves. In view of these abnormalities, and since tests for gluten
sensitivity had not been performed during the earlier
investigations, the possibility of gluten sensitivity was
considered. A comprehensive IgG and IgA panel was ordered against a
repertoire of wheat proteins and peptides, as well as against tTg
and various tissue antigens. This comprehensive gluten sensitivity
and immune reactivity screen revealed IgG against wheat antigens,
.alpha.-gliadin 33- and 17-mer, .gamma.- and .omega.-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.
[0079] Furthermore, antibodies against ganglioside, cerebellar,
synapsin, myelin basic protein, collagen, thyroglobulin and thyroid
peroxidase were tested, 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
methylprednisolone was implemented, and gradually the sensory,
motor and visual symptoms improved. In addition, based on the very
high levels of IgG and some IgA antibodies against a repertoire of
wheat antigens and peptides, a gluten-free diet was introduced, and
12 weeks later marked improvement was observed in the patient's
clinical symptomotology. She continued the 100% gluten-free diet
under the observation of a dietitian, and the steroid treatment was
stopped. Six months after introduction of the diet antibody tests
against wheat antigens, peptides, and human tissue were repeated;
more than 60% reduction in antibody levels was observed, and the
patient became almost asymptomatic.
[0080] Discussion: From this data I have concluded that a patient
may suffer from gluten immune reactivity and sensitivity without
having abnormal tissue histology or flat erosive gastritis and
antibody against tTg based on which a diagnosis of celiac disease
is normally made. If patients 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 of wheat
proteins and peptides, patients' symptomatologies may mislead many
clinicians into treating their patients for lupus, MS-like
syndrome, neuromyeltitis optica, and many other 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 so that
intervention with a gluten-free diet will be instrumental in
reversing the autoimmune conditions associated with gluten immune
reactivity and sensitivity. Otherwise, untreated and/or mistreated,
the patient will develop multiple autoimmune disorders.
[0081] D. Case Report #4: Gluten Immune Reactivity and Sensitivity
Overlapping with Crohn's Disease
[0082] 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 dysfunctional responses against them, is believed
to lead to dysregulated immunity, altered intestinal barrier
function, and possibly autoimmunity (16).
[0083] Here, a 32 year-old man presented with gastrointestinal
discomfort and diarrhea 2-3 times per month. Laboratory results
including chemistry panel, CBC, iron, ferritin, transferrin,
vitamin B-12, thyroid function, 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 examinations. These tests were
microbiological evaluation of the stool and blood tests for
antibodies against H. pylori, Saccharomyces and gliadin. Stool
testing with respect to the detection of Salmonella, 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/mL (normal values=<20 U/mL). 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. Three years later after seeing
the frequency of the watery diarrhea 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 specialist for a second opinion. Gastric
and duodenal biopsies and endoscopy 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. D-xylose absorption test was performed; the resulting
value of 1.89 g/5 h in urine was suggestive of malabsorption.
Immunoserologically ANA titers were below 1:40, p-ANCA and c-ANCA
were negative, but the IgA anti-Saccharomyces antigen (ASCA) was
positive at 85 U/mL (normal=<10 U/mL). Based on the increased
frequency 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 initiated but the
frequency of the diarrhea remained unchanged. In addition the
patient was treated with 230 mg of methylprednisolone, and
2.times.1000 mg of mesalazine. Two years after this treatment the
patient developed entero-enteric fistulae in the terminal ileum
with sigmoid affection. After admission to the hospital,
ileocolectomy was performed and 22 cm of the ileum was resected.
Upon his release remission maintenance with 3.times.500 mg of
mesalazine was implemented.
[0084] 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 treatment attempts were made using aspirin, loperamide,
and budesonide, unfortunately without significant clinical
improvement. Furthermore, the patient was losing more weight on a
monthly basis. A complete review of the medical history revealed
the fact that almost thirteen years earlier, gliadin 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, a comprehensive
test for the assessment of gluten immune reactivity and sensitivity
was ordered. This included IgG and IgA against wheat, native and
deamidated .alpha.-gliadin peptides, .gamma.-gliadin,
.omega.-gliadin, glutenin, gluteomorphin, prodynorphin, gliadin-tTg
complex, transglutaminase, wheat germ agglutinin, and GAD-65.
[0085] Results depicted in Tables 6 and 7, Sample #3 show that the
patient had a significant elevation of IgG antibodies against 11
out of 12 tested antigens, and IgA antibodies against wheat,
.alpha.-gliadin 33-mer, .omega.-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
diagnosis of gluten sensitivity was also made. A diet consisting of
rice, potato, and other gluten-free/yeast-free foods was commenced
immediately, which led after six weeks to a complete cessation of
diarrhea. Upon continuation of the gluten-free diet, not only did
stool consistency become normal but the patient also started
gaining 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.
[0086] 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 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
autoimmune disorders was published in 1999 (Ventura A et al.,
Gastroenterol, 117: 303-310, 1999); unfortunately, this was
ignored.
[0087] It is contemplated herein that continuous exposure to
environmental factors such as wheat antigen induced inflammation
for a prolonged period of time, resulting in inflammatory bowel
disease or Crohn's disease.
Conclusions Regarding Case Reports
[0088] 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 erroneous 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. 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).
FIG. 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 information and confirmation of celiac disease, Crohn's
disease, gluten immune reactivity/sensitivity and autoimmunity.
[0089] It should be apparent to those skilled in the art that many
more modifications besides those already described are possible
without departing from the inventive concepts herein. The inventive
subject matter, therefore, is not to be restricted 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 possible manner consistent 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 combined with
other elements, components, or steps that are not expressly
referenced. Where the specification claims refers to at least one
of something 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.
REFERENCES
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S A, Watson R G, et al. Malignancy and mortality in a
population-based cohort of patients with coeliac disease or `gluten
sensitivity.` World J Gastroenterol, 13: 146-151, 2007. [0092] 3.
Tanabe S. Analysis of food allergen structures and development of
foods for allergic patients. Biosci Biotechnol Biochem, 72:
649-659, 2008. [0093] 4. Chin R L, Latov N, Green P H R, et al.
Neurological complications of celiac disease. J Clin Neuromusc Dis,
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Sapone A, Lammers K M, Casolaro V, et al. Divergence of gut
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Kellermann G H. The immunology of immediate and delayed
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[0103] 14. Jacob S et al. Gluten sensitivity and neuromyelitis
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1028-1030, 2005. [0104] 15. Egan C E, et al. Synergy between
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658-670, 2011. [0105] 16. Kaser A et al. Inflammatory Bowel
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and Villanaci V. Celiac disease: diagnostic criteria in progress.
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characterization of the repertoire of wheat antigens and peptides
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sensitivity and Crohn's disease. ISRN Allergy. Article ID 950104,
1-12.
TABLE-US-00002 [0107] TABLE 1 Reportable Ranges & Reference
Ranges (ELISA Index) Reportable In Range Out of Range Reference
Antigens Range (Negative) Equivocal (Positive) Range Wheat IgG
0.3-6.4 0.24-1.0 1.01-1.3 >1.3 0.24-1.3 Wheat IgA 0.4-9.7
0.0-1.9 1.91-2.4 >2.4 0.0-2.4 .alpha.-Gliadin-33 IgG 0.3-10.0
0.16-1.1 1.11-1.4 >1.4 0.16-1.4 .alpha.-Gliadin-33 IgA 0.6-16.0
0.6-1.5 1.51-1.8 >1.8 0.6-1.8 .alpha.-Gliadin-17 IgG 0.3-5.0
0.0-1.1 1.11-1.5 >1.5 0.0-1.5 .alpha.-Gliadin-17 IgA 0.6-14.8
0.4-1.6 1.61-2.0 >2.0 0.4-2.0 .gamma.-Gliadin-15 IgG 0.4-12.0
0.14-1.3 1.31-1.7 >1.7 0.14-1.7 .gamma.-Gliadin-15 IgA 0.5-16.0
0.7-1.6 1.61-1.9 >1.9 0.7-1.9 .omega.-Gliadin IgG 0.5-1.0
0.24-1.3 1.31-1.6 >1.6 0.24-1.6 .omega.-Gliadin IgA 0.6-15.4
0.6-1.5 1.51-1.8 >1.8 0.6-1.8 Glutenin IgG 0.2-6.0 0.0-1.1
1.11-1.5 >1.5 0.0-1.5 Glutenin IgA 0.5-15.9 0.5-1.4 1.41-1.7
>1.7 0.5-1.7 Gluteomorphin IgG 0.3-8.0 0.12-1.2 1.21-1.5 >1.5
0.12-1.5 Gluteomorphin IgA 0.5-15.1 0.6-1.5 1.51-1.8 >1.8
0.6-1.8 Prodynorphin IgG 0.4-13.0 0.13-1.7 0.131-1.7 >1.7
0.13-1.7 Prodynorphin IgA 0.6-16.0 0.6-1.5 1.51-1.8 >1.8 0.6-1.8
Gliadin-tTg Complex IgG 0.4-13.0 0.12-1.2 1.21-1.6 >1.6 0.12-1.6
Gliadin-tTg Complex IgA 0.6-15.5 0.5-1.3 1.31-1.6 >1.6 0.5-1.6
Transglutaminase IgG 0.5-12.0 0.21-1.1 1.11-1.4 >1.4 0.21-1.4
Transglutaminase IgA 0.5-10.9 0.6-1.3 0.31-1.5 >1.5 0.6-1.5
Wheat Germ Agglutinin IgG 0.5-10.0 0.17-1.1 1.1-1.5 >1.5
0.17-1.5 Wheat Germ Agglutinin IgA 0.6-16.9 00.9-1.6 1.61-1.9
>1.9 0.9-1.9 Glutamic Acid Decarboxylase IgG 0.4-12.0 0.29-1.1
1.11-1.3 >1.3 0.29-1.3 Glutamic Acid Decarboxylase IgA 0.8-12.9
0.6-1.3 1.31-1.5 >1.5 0.6-1.5
TABLE-US-00003 TABLE 2 IgG Antibody Pattern of 3 Patients with
Celiac Disease reacting against various Wheat Antigens, Peptides
and Tissue Antigens expressed as Optical Density with Calculation
of Indices *Alpha Alpha Gamma Omega Gliadin- Wheat Wheat Gliadin
Gliadin Gliadin Gliadin Glutenin Gluteo- Pro- tTg Germ Antigens
33-mer 17-mer 15-mer 17-mer 21-mer morphin Dynorphin Complex **tTg
Agglutinin ***GAD-65 Cal 1 0.41 0.33 0.35 0.32 0.51 0.40 0.50 0.38
0.39 0.46 0.37 0.38 Cal 2 0.44 0.35 0.35 0.32 0.25 0.43 0.49 0.37
0.40 0.44 0.36 0.42 Sample 1 3.77 1.04 3.86 1.02 3.84 1.34 1.44
3.84 1.76 3.83 1.15 3.81 (OD) 3.78 1.08 3.85 1.06 3.85 1.28 1.45
3.84 1.73 3.85 1.20 3.81 Index 8.91 3.11 11.02 3.26 10.14 3.19 2.92
10.32 4.43 8.55 3.24 9.52 Sample 2 3.85 3.87 1.13 1.10 3.86 1.08
3.84 1.26 1.37 3.84 1.45 0.98 (OD) 3.85 3.86 1.11 1.17 3.84 1.09
3.84 1.31 1.36 3.84 1.33 1.04 Index 9.09 11.32 3.21 3.55 10.15 2.63
7.76 3.44 3.46 8.55 3.83 2.53 Sample 3 3.77 0.89 1.11 3.53 1.23
3.37 1.31 1.15 3.84 1.37 3.84 3.54 (OD) 3.79 0.80 1.00 3.51 1.15
3.39 1.21 1.09 3.85 1.28 3.83 3.54 Index 8.93 2.46 3.03 11.05 3.14
8.23 2.55 2.99 9.74 2.95 10.56 8.85 *Native + Deamidated Gliadin
33-mer **Transglutaminase ***Glutamic Acid Decarboxylase Index =
Mean OD of patients Mean OD of calibrators ##EQU00002##
TABLE-US-00004 TABLE 3 IgA Antibody Pattern of 3 Patients with
Celiac Disease reacting against various Wheat Antigens, Peptides
and Tissue Antigens expressed as Optical Density with Calculation
of Indices *Alpha Alpha Gamma Omega Gliadin- Wheat Wheat Gliadin
Gliadin Gliadin Gliadin Glutenin Gluteo- Pro- tTg Germ Antigens
33-mer 17-mer 15-mer 17-mer 21-mer morphin Dynorphin Complex **tTg
Agglutinin ***GAD-65 Cal 1 0.36 0.37 0.36 0.36 0.31 0.41 0.43 0.39
0.39 0.37 0.41 0.48 Cal 2 0.39 0.37 0.36 0.36 0.39 0.41 0.43 0.41
0.39 0.38 0.39 0.39 Sample 1 3.00 3.64 1.23 3.82 0.47 3.82 3.83
3.39 3.83 3.84 3.87 0.83 (OD) 3.03 3.56 1.22 3.81 0.45 3.81 3.82
3.40 3.81 3.86 3.86 0.84 Index 7.97 9.69 3.43 10.67 1.31 9.42 8.88
8.51 9.84 10.29 9.69 1.93 Sample 2 3.55 0.96 3.83 0.95 3.82 1.75
1.31 3.82 3.87 3.85 1.65 3.80 (OD) 3.53 1.01 3.82 0.99 3.83 1.97
1.40 3.83 3.87 3.86 1.77 3.83 Index 9.34 2.65 10.69 2.71 10.96 4.59
3.14 9.60 9.98 10.30 4.28 8.83 Sample 3 3.81 3.84 2.99 3.83 0.99
0.40 1.85 1.33 3.83 3.85 3.81 3.48 (OD) 3.80 3.85 2.92 3.81 0.94
0.35 1.74 1.15 3.82 3.84 3.82 3.43 Index 10.05 10.36 8.27 10.68
2.76 0.92 4.16 3.11 9.86 10.26 9.56 7.99 *Native + Deamidated
Gliadin 33-mer **Transglutaminase ***Glutamic Acid Decarboxylase
Index = Mean OD of patients Mean OD of calibrators ##EQU00003##
TABLE-US-00005 TABLE 4 IgG Antibody Pattern of 3 Patients with
Gluten Immune Reactivity/Sensitivity/Autoimmunity reacting against
various Wheat Antigens, Peptides and Tissue Antigens expressed as
Optical Density with Calculation of Indices *Alpha Alpha Gamma
Omega Gliadin- Wheat Wheat Gliadin Gliadin Gliadin Gliadin Glutenin
Gluteo- Pro- tTg Germ Antigens 33-mer 17-mer 15-mer 17-mer 21-mer
morphin Dynorphin Complex **tTg Agglutinin ***GAD-65 Cal 1 0.33
0.42 0.34 0.33 0.38 0.38 0.36 0.35 0.33 0.35 0.35 0.36 Cal 2 0.36
0.48 0.36 0.38 0.42 0.44 0.39 0.40 0.45 0.38 0.38 0.43 Sample 1
3.86 0.95 3.22 1.22 0.41 1.29 1.54 3.81 0.67 0.44 1.28 1.25 (OD)
3.87 0.88 3.26 1.26 0.33 1.34 1.36 3.84 0.64 0.41 1.31 1.20 Index
11.14 2.03 9.37 3.46 0.93 3.21 3.84 10.31 1.66 1.17 3.55 3.09
Sample 2 3.88 3.87 2.71 2.05 3.86 1.98 1.97 1.95 0.41 0.40 1.95
2.14 (OD) 3.88 3.87 2.65 2.10 3.85 2.28 2.20 2.19 0.37 0.41 2.18
2.21 Index 11.18 8.58 7.75 5.80 9.72 5.21 5.52 5.58 1.00 1.12 5.66
5.50 Sample 3 3.85 2.44 2.38 3.86 2.41 2.50 3.86 2.49 1.49 1.00
3.86 3.88 (OD) 3.85 2.36 2.33 3.86 2.40 2.40 3.86 2.40 1.58 0.96
3.86 3.87 Index 11.10 5.33 6.81 10.79 6.05 5.99 10.23 6.60 3.91
2.69 10.59 9.79 *Native + Deamidated Gliadin 33-mer
**Transglutaminase ***Glutamic Acid Decarboxylase Index = Mean OD
of patients Mean OD of calibrators ##EQU00004##
TABLE-US-00006 TABLE 5 IgA Antibody Pattern of 3 Patients with
Gluten Immune Reactivity/Sensitivity/Autoimmunity reacting against
various Wheat Antigens, Peptides and Tissue Antigens expressed as
Optical Density with Calculation of Indices *Alpha Alpha Gamma
Omega Gliadin- Wheat Wheat Gliadin Gliadin Gliadin Gliadin Glutenin
Gluteo- Pro- tTg Germ Antigens 33-mer 17-mer 15-mer 17-mer 21-mer
morphin Dynorphin Complex **tTg Agglutinin ***GAD-65 Cal 1 0.392
0.462 0.401 0.368 0.400 0.449 0.467 0.445 0.400 0.430 0.485 0.397
Cal 2 0.421 0.462 0.414 0.379 0.418 0.453 0.479 0.481 0.392 0.402
0.455 0.404 Sample 1 2.176 0.371 0.995 3.098 0.346 2.240 2.141
2.452 0.271 0.254 2.470 0.664 (OD) 2.248 0.424 0.827 3.123 0.407
2.402 2.297 2.531 0.329 0.245 2.354 0.724 Index 5.442 0.860 2.236
8.328 0.921 5.146 4.691 5.381 0.758 0.600 5.132 1.733 Sample 2
1.763 1.442 0.260 1.332 0.377 1.073 1.007 0.994 0.195 0.343 1.101
0.743 (OD) 1.664 1.573 0.252 1.483 0.342 1.063 1.058 1.089 0.205
0.351 1.144 0.764 Index 4.215 3.263 0.628 3.768 0.879 2.368 2.183
2.249 0.505 0.834 2.388 1.881 Sample 3 3.862 0.429 0.350 0.208
0.364 0.269 0.401 0.336 0.335 0.438 3.868 0.359 (OD) 3.828 0.426
0.397 0.245 0.325 0.290 0.546 0.372 0.360 0.473 3.833 0.438 Index
9.459 0.925 0.917 0.606 0.842 0.620 1.001 0.765 0.878 1.095 8.193
0.995 *Native + Deamidated Gliadin 33-mer **Transglutaminase
***Glutamic Acid Decarboxylase Index = Mean OD of patients Mean OD
of calibrators ##EQU00005##
TABLE-US-00007 TABLE 6 IgG Antibody Pattern of 3 Patients with
Crohn's Disease reacting against various Wheat Antigens, Peptides
and Tissue Antigens expressed as Optical Density with Calculation
of Indices *Alpha Alpha Gamma Omega Gliadin- Wheat Wheat Gliadin
Gliadin Gliadin Gliadin Glutenin Gluteo- Pro- tTg Germ Antigens
33-mer 17-mer 15-mer 17-mer 21-mer morphin Dynorphin Complex **tTg
Agglutinin ***GAD-65 Cal 1 0.45 0.41 0.38 0.39 0.36 0.39 0.55 0.47
0.71 0.49 0.41 0.55 Cal 2 0.38 0.33 0.44 0.44 0.37 0.40 0.54 0.56
0.60 0.49 0.48 0.46 Sample 1 3.74 1.56 0.65 2.29 0.56 2.10 0.54
0.69 3.81 0.81 1.23 1.83 (OD) 3.68 1.59 0.56 2.15 0.57 2.01 0.51
0.55 3.54 0.75 1.25 1.80 Index 8.97 4.26 1.47 5.30 1.54 5.22 0.96
1.19 5.59 1.59 2.79 3.59 Sample 2 3.87 0.47 0.72 0.55 0.59 0.49
0.52 0.44 1.04 0.76 3.83 3.87 (OD) 3.84 0.43 0.61 0.53 0.46 0.48
0.51 0.37 1.11 0.70 3.80 3.86 Index 9.31 1.22 1.62 1.29 1.43 1.23
0.94 0.78 1.63 1.49 8.62 7.63 Sample 3 3.86 3.79 3.86 3.67 3.85
3.24 3.84 3.86 1.71 3.80 3.82 3.84 (OD) 3.84 3.79 3.84 3.59 3.85
3.25 3.83 3.83 1.73 3.74 3.80 3.59 Index 9.31 10.23 9.33 8.67 10.51
8.22 7.03 7.43 2.62 7.71 8.60 7.34 *Native + Deamidated Gliadin
33-mer **Transglutaminase ***Glutamic Acid Decarboxylase Index =
Mean OD of patients Mean OD of calibrators ##EQU00006##
TABLE-US-00008 TABLE 7 IgA Antibody Pattern of 3 Patients with
Crohn's Disease reacting against various Wheat Antigens Peptides
and Tissue Antigens expressed as Optical Density with Calculation
of Indices *Alpha Alpha Gamma Omega Gliadin- Wheat Wheat Gliadin
Gliadin Gliadin Gliadin Glutenin Gluteo- Pro- tTg Germ Antigens
33-mer 17-mer 15-mer 17-mer 21-mer morphin Dynorphin Complex **tTg
Agglutinin ***GAD-65 Cal 1 0.37 0.39 0.40 0.39 0.43 0.43 0.46 0.47
0.41 0.42 0.48 0.42 Cal 2 0.45 0.44 0.46 0.42 0.47 0.51 0.54 0.50
0.48 0.48 0.49 0.47 Sample 1 3.81 3.80 0.22 3.68 0.26 3.13 2.55
2.70 0.97 0.93 3.81 1.03 (OD) 3.82 3.79 0.20 3.17 0.27 3.18 2.50
2.58 1.00 0.97 3.80 1.02 Index 9.30 9.08 0.49 8.50 0.59 6.68 5.06
5.44 2.23 2.13 7.85 2.32 Sample 2 3.00 0.87 0.67 0.74 0.33 0.66
1.43 1.12 0.98 0.29 1.00 1.07 (OD) 3.43 0.85 0.64 0.70 0.32 0.66
1.12 1.03 1.00 0.31 1.02 1.10 Index 7.85 2.05 1.52 1.79 0.72 1.40
2.56 2.21 2.23 0.67 2.08 2.43 Sample 3 3.89 3.56 0.99 0.99 2.25
1.05 1.12 3.85 1.00 1.18 3.73 3.87 (OD) 3.89 3.48 0.99 0.99 2.25
1.03 1.10 3.82 0.99 1.20 3.83 3.85 Index 9.48 8.43 2.27 2.45 4.98
2.20 2.23 7.90 2.25 2.65 7.79 8.69 *Native + Deamidated Gliadin
33-mer **Transglutaminase ***Glutamic Acid Decarboxylase Index =
Mean OD of patients Mean OD of calibrators ##EQU00007##
TABLE-US-00009 TABLE 8 IgG and IgA Reactivity For Detection Of
Gluten Immune Reactivity And Sensitivity, And Its Differentiation
From Celiac Disease INDICATIONS Testing Testing Proposed for
Proposed for Gluten Gluten Immune Testing Immune Reactivity,
Proposed for Reactivity and Sensitivity and Current Proposed Celiac
Disease Sensitivity in Autoimmunity Antigens and Antibody Testing
for Testing for in this Patent this Patent in this Patent Peptides
Isotype Celiac Disease Celiac Disease Application Application
Application .alpha.-Gliadin 33-Mer IgG + or - + or - + or - + + IgA
+ + + + or - + or - Deamidated IgG + or - + or - + or - - -
.alpha.-Gliadin 33-Mer IgA + + + - - Transglutaminase IgG + or - +
or - + or - + or - + or - IgA + + + - - Gliadin IgG Not + or - + or
- + or - + or - Transglutaminase IgA tested + + - - Complex
.alpha.-Gliadin 17-Mer IgG Not Not + or - + + IgA tested tested + +
or - + or - .gamma.-Gliadin 15-Mer IgG Not Not + or - + + IgA
tested tested + + or - + or - .omega.-Gliadin 17-Mer IgG Not Not +
or - + + IgA tested tested + + or - + or - Glutenin 21-Mer IgG Not
Not + or - + + IgA tested tested + + or - + or - Deamidated IgG Not
Not + or - - - Glutenin 21-Mer IgA tested tested + - -
Gluteomorphin IgG Not Not + or - + + IgA tested tested + + or - +
or - Prodynorphin IgG Not Not + or - + + IgA tested tested + + or -
+ or - Wheat Germ IgG Not Not + or - + + Agglutinin IgA tested
tested + + or - + or - Glutamic Acid IgG Not Not + or - + or - +
Decarboxylase tested tested Cerebellar Peptide IgG Not tested Not
tested + or - + or - + Other Tissue IgG Not Not + or - + or - +
Antigens tested tested
* * * * *