Note: Descriptions are shown in the official language in which they were submitted.
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IMMZTNOLOGICAL ASSAY FOR DETECTION OF ANTIBODIES IN
CELIAC DISEASE
This application claims the priority of U.S.
provisional application 60/160,548 filed on October 20,
1999, the disclosure of which is incorporated herein by
reference.
BACKGROUND OF THE INVENTION
Field of the Invention
The present invention relates generally to the
field of celiac disease. More particularly, the present
invention provides a sensitive immunological assay for
the detection of antibodies implicated in celiac
disease.
Description of Related Art
Celiac disease (CD) is a disease of the intestinal
mucosa and is usually manifested in infants and
children. CD is associated with an inflammation of the
mucosa, which causes malabsorption. Individuals with
celiac disease do not tolerate a protein called gluten,
which is present in wheat, rye, barley and possibly
oats. When exposed to gluten, the immune system of an
individual with CD responds by attacking the lining of
the small intestine. The only treatment of CD is a
gluten-free diet, which usually results in morphological
and clinical improvement.
Currently, the routine procedure to detect celiac
disease is intestinal biopsy for checking damage to the
intestinal lining. Recently, it has been reported that
antibodies directed against gliadin, endomysial antigen
(EMA), or reticulin can be detected in CD. Thus, ELISAs
for gliadin and immunofluorescence assays for EMA and
reticulin have been suggested for the diagnosis of CD.
Further, since transglutaminase (tTG) has been
identified as the endomysial antigen involved in CD,
immunological assays are being proposed to detect
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antibodies using tTG as the antigen (Schuppan et al., WO
98/03892, 1998).
Gliadins are a class of proteins that can be
isolated from wheat. These include the alpha, gamma,
epsilon, delta and omega gliadins. Antibodies to
gliadins have been reported in CD. Thus,
immunoreactivity is observed against the gliadins as
well as against glutenin, which is a partially purified
fraction of wheat and contains gliadins.
Transglutaminases (EC 2.3.2.13) are a diverse
family of Ca2+ dependent enzymes that are highly
ubiquitous and highly conserved across species.
Transglutaminases catalyze the covalent cross-linking of
specific proteins through the formation of isopeptide
bonds between a-carboxyl groups of glutamine residues in
one polypeptide and E-NHz groups of lysine residues in
another. The resulting polymer network is stable and
resistant or proteolysis, increasing the resistance of
tissue to chemical, enzymatic and mechanical disruption.
Of all the transglutaminases, tissue transglutaminases
(tTG) is the most widely distributed. tTG provides the
focus of the autoimmune response in CD, and has
therefore been used for diagnosis of CD.
Although immunoassay techniques have been described
for gladins and tTG for the diagnosis of CD, these
assays do not detect all cases of celiac disease or
sprue that are detected by intestinal biopsy. As a
result, there is an ongoing need for the development of
sensitive non-invasive tests for the diagnosis of celiac
disease.
SUMMARY OF THE INVENTION
The present invention discloses a method for the
diagnosis of celiac disease that is more sensitive that
the methods of the prior art. The present method
comprises detecting antibodies in the sera of patients
to a combination of transglutaminase and its substrate
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such as gliadin, glutenin or other peptides having
multiple glutamines. The sensitivity of the method
detecting antibodies to a combination of tTG and its
substrates is greater than the additive sensitivity of
detecting antibodies to tTG alone or its substrates
alone.
Accordingly, it is an object of the present
invention to provide a sensitive method for the
diagnosis of celiac disease.
It is another object of the present invention to
provide a method of diagnosing celiac disease comprising
the step of detecting the presence of antibodies to the
combination of tTG and one or more substrates of tTG.
DESCRIPTION OF PREFERRED EMBODIMENTS
The term "substrate of tTG" or "tTG substrate" as
used herein for the purposes of specification and claims
means a peptide or polypeptide, irrespective of whether
or not it is an enzymatic substrate of tTG, that
interacts with tTG such that hidden epitopes on the
substrate or tTG are exposed, the exposure of the hidden
epitopes being detected as a synergistic increase in the
detection of antibodies. An example of a substrate of
tTg is a peptide or polypeptide having multiple
glutamines such as polyglutamine, protamine sulfate or
natural substrates such as gliadins and glutenin.
The present invention relates to increasing the
sensitivity of diagnosis of celiac disease by detecting
the presence of antibodies to tTG in the presence of its
substrate. It was unexpectedly observed that when tTG
was allowed to interact with a substrate prior to
contact with the sample containing antibodies, the
sensitivity of detection increased synergistically.
In one embodiment, the substrate of tTG was
gliadin. That the increased sensitivity was not simply
due to the detection of antibodies to tTG and to gliadin
was demonstrated by the synergistic increase in the
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detection. Thus, more samples were detected as positive
when tTG was allowed to react with its substrate
(gliadin) prior to contact with the antibodies, than
when either tTG alone or gliadin alone was used as the
antigen. Further, when tTG was incubated with a
substrate not known to be a natural substrate, i.e.,
protamine sulfate, a similar increase in sensitivity was
observed. Although not intending to be bound by any
particular theory, it is considered that tTG undergoes
conformational change upon interacting with a substrate
and in doing so, exposes epitopes that are relevant in
vivo and that are not available without the substrate.
For the method of the present invention, the
substrate of tTG is coated on to a solid matrix. Any
standard immunoassay solid matrix such as a microtitre
plate, beads and the like can be used. Transglutaminase
(tTG) can be purified from any source such as murine,
porcine, equine, human, monkey etc., or can be prepared
by recombinant technology. tTG is also available
commercially. Substrates for tTG are, or contain,
peptides or polypeptides having multiple glutamine
residues. Suitable substrates for tTG may be natural
substrates such as gliadins or glutenin, or substrates
not known to be natural substrates like protamine
sulfate or polyglutamine. When the substrates of tTG
is soluble only in an acidic medium, as is the case with
gliadins and glutenin, it is preferable to coat a solid
matrix first with the substrate in an acidic medium such
as acetic acid. After a suitable period of coating,
excess acid is washed away and then tTG is added. In
the case of substrates that do not require an acidic
medium for solubilization, such as protamine sulfate,
substrate and tTg may be coated on a solid matrix in any
order or the two may be incubated together in aqueous
solution and then coated on to a solid matrix.
For example, in one embodiment, the solid matrix
can be coated with gliadin, glutenin or both. In a
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preferred embodiment, the gliadin is gamma gliadin.
Determination of appropriate concentrations of the
substrate and tTG are well within the purview of one
skilled in the art. In one embodiment, gliadin and
glutenin were used in the range of 250ng/ml to 3~,g/ml.
Next, the solid matrix can be coated with tTG. In one
embodiment, the concentration of tTG for coating was
5ug/ml to 20ug/ml. After removal of unbound antigens,
nonspecific binding sites on the solid matrix can be
blocked by methods well known in the art. The tTG plus
substrate coated solid matrix can be used immediately
for testing patient sera, or can be stored up to 2
years.
Antibodies associated with celiac diseases can be
detected in patient sera using the solid matrix as
coated above. Briefly, serum at a suitable dilution is
incubated with the solid matrix coated with tTG and its
substrate. Unbound materials are removed using standard
techniques and bound antibodies are detected and
quantitated using enzyme linked or fluorescent detection
agents. These techniques are well known in the art of
immunoassays. For example, for detection of antibodies
of human origin, an anti-human IgG, IgM or IgA or
antigen binding fragments thereof having a detectable
label may be used. The detectable label may be an
enzyme including, but not limited to, alkaline
phosphatase, ~i-lactamase, ~-galactosidase, urease or
horseradish peroxidase. In a preferred embodiment, the
label is alkaline phosphatase or horseradish peroxidase.
Suitable substrates for these enzymes are well known in
the art and include p-nitrophenyl-phosphate, 5-bromo-4-
chloro-3-indolyl-phospate, 3,3-diaminobenzidine, and -
phenylenediamine.
The presence of antibodies to tTG plus its
substrate is considered to be an indication of the
presence of CD.
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The following examples are provided to illustrate
the invention and are not intended to be restrictive.
Example 1
This embodiment illustrates the coating of a solid
matrix with the antigens (tTG plus substrates) and
detection of specific antibodies using the antigen-
coated solid matrix. In this illustration, prior to
coating the plates with tTG, the microtitre plates are
coated with glutenin and gliadin (Y-gliadin). Both
glutenin and gliadin were dissolved in 1 mM acetic acid
(57 ~.1 glacial acetic acid in 1 L distilled water). at
2.5~.g/ml concentration and mixed to make a uniform
solution. One hundred microliters of the glutenin-gamma
gliadin mixture were added to each of the microtitre
well. Plates were incubated at 2-8°C overnight. Next,
the plates were coated with 100~C1 of tTG solution,
prepared at a concentration of 10~.g/ml in a coating
buffer (7.88 g of Tris-HC1, 8.78 of NaCl, and 0.7358 of
calcium chloride dihydrate in 1 L H20). The plates were
incubated overnight at 2-8°C, washed four times with tTG
wash buffer (7.888 of Tris-HC1, 8.768 of NaCl, 3.728 of
EDTA disodium salt, 1 mM Tween 20 in 1 L Hz0). The
washed plates were incubated with blocking buffer (lo
calf serum in PBS, 0.090 sodium azide). After blocking,
the plates were washed with tTG wash buffer and dried.
Patient serum was diluted 1:50 in serum diluent (1% calf
serum in PBS) and 100 u1 of the diluted patient serum
was added to the plates and the plates were incubated
for 1 hour at room temperature (RT). Unbound materials
were removed by washing with PBS. Alkaline phosphatase
conjugated anti-human IgA was added to the plates for
about 30 minutes. Unbound materials were removed by
washing. An alkaline phosphatase substrate was added to
develop color, and detection and quantitation was
performed using a spectrophotometer.
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The results of an experiment, wherein detection of
antibodies was carried out with plates coated with i)
tTG, glutenin and gliadin, ii) only glutenin and
gliadin, and iii) only tTG are presented below in Table
1
Table 1
Sera ID tTG, gliadin glutenin tTG EMA
and glutenin and gliadin coated titre
coated coated (EU/ml)
(EU/ml) (EU/ml)
Col. A Col. B Col. C Col. D Col. E
98-5901 198.8* 21.0* 33.4* 640*
97-2043 28.7* 5.4 9.7 160*
97-4684 64.8* 6.5 8.0 320*
97-2873 63.4* 6.5 25.4* 160*
97-6405 49.0* 16.2* 0.0 160*
97-2449 35.4* 2.2 0.0 320*
98-1537 68.8* 7.8 19.8 160*
The cutoff value for each assay is 20EU/ml. Values
that are above the cutoff value are positive and are
indicated by an asterisk. Column A indicates the sera
identity. Column E indicates that the sera selected for
this analysis are all anti-endomysial antigen positive
(Gold Standard diagnostic criteria) as determined by
immunofluorescence assay. Column B shows the result of
the values obtained by using the method of the present
invention. As described above, this method includes
pre-coating of the plates with glutenin and gamma
gliadin followed by the addition of tTG. In contrast,
when the same samples were tested using plates coated
with tTG alone (column D) or with glutenin and gamma
gliadin, without tTG (column C), most of the values were
below the cutoff value and hence would be deemed
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negative. While not intending to be bound by any
particular theory, it is considered that the conditions
of column B, allows the tTG to interact with its
substrate, gliadin and glutenin, thereby eliciting the
Celiac Disease specific epitopes on tTG that can be
efficiently recognized by the method of the present
invention.
Example 2
This embodiment demonstrates that increased
sensitivity is achieved by using a single substrate of
tTG. To illustrate this embodiment, the detection was
carried out essentially as described in Example 1 for
samples that were positive using a combination of
glutenin and Y-gliadin. Briefly, microtitre plates were
coated with a-gliadin, y-gliadin or glutenin. After
washing to remove unbound materials, the plates were
incubated with tTG. After incubation, plates were
washed and an aliquot of test samples was added to the
wells. Color development was carried out by alkaline
phosphatase labeled anti-human IgA followed by the
addition of a substrate for alkaline phosphatase. Color
was quantitated in a spectrophotometer. Samples with a
reading of 3 Standard deviations above the control were
marked as positive. The results from a representative
sample are presented in Table 2.
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Table 2
Sample a-gliadin y-gliadin glutenin y-gliadin +
glutenin
98-6317 positive positive positive positive
98-3810 positive positive ND positive
98-840 positive positive positive positive
98-6316 positive positive positive positive
98-5798 ND positive positive positive
97-2101 positive positive positive positive
97-6719 positive positive positive positive
97-49 positive ND ND positive
97-1048 positive positive positive positive
1~ inaicates not aeterminea.
These results demonstrate that using a single
substrate such as a- or y-gliadin or the partially
purified fraction glutenin, which contains gliadins, is
equally effective for the method of the present
invention.
Example 3
This embodiment demonstrates that to increase
sensitivity of the assay, tTG can be incubated with a
natural substrate as described above or a substrate not
known to be a natural substrate but which contains
multiple glutamine residues such as protamine sulfate.
or polyglutamine. To illustrate this embodiment, tTG
was incubated with protamine sulfate essentially as
described in Example 1. Briefly, microtiter plates were
coated with 50ug/ml solution of protamine sulfate for 2
hours at room temperature. Plates were washed with PBS
and incubated with lug/well tTG for 1 hour at room
temperature. Plates were washed with tTG wash buffer,
blocked with O.lo Triton-x-100 in PBS, washed three
times with tTG wash buffer. Serum samples from patients
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that were known to be positive from EMA titers were then
added to the wells. After incubation for 1 hour at room
temperature, plates were washed and alkaline phophatase
conjugated anti-human IgA was added. Following
incubation for 30 minutes are room temperature, the
plates were washed and the substrate for alkaline
phosphatase was added. Color was quantitated in a
spectrophotometer. The results are presented in Table
3.
Table 3
SAMPLE tTG + protamine tTG + glutenin +
sulfate Y-gliadin
00-3638 positive positive
98-1002 positive positive
98-5901 positive positive
97-2873 positive positive
97-1062 positive positive
97-6405 positive positive
97-3082 positive positive
These results indicate that all samples that were
positive using tTG plus glutenin and gliadin, were also
found to be positive with tTG plus protamine sulfate.
Thus, both natural substrates of tTG and other peptides
or polypeptides containing multiple glutamine residues
can be used for the method of the present invention.
In a variation of this illustration, plates were
coated with protamine sulfate, and then blocked with
blocking buffer containing 0.1o triton x-100 in PBS
prior to being coated with tTG and the plates were
washed with tTG wash buffer. Detection was carried out
as above. No difference was observed between blocking
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first and then incubating with tTG and incubating with
tTG and then blocking.
These data indicate that the increased sensitivity
observed in the present invention is achieved by
allowing tTG to interact with its natural or unnatural
substrates. While not intending to be bound by any
particular theory, it is considered that tTG interacting
with its substrate more accurately represents the
situation in vivo and therefore, the sensitivity of the
assay is closer to the sensitivity of the
immunofluorescence detection techniques for the presence
of EMA immunoreactivity in intestinal biopsied sections.
Increasing the sensitivity of detection will help in the
diagnosis of celiac disease without the necessity of a
biopsy.
From the foregoing, it will be obvious to those
skilled in the art that various modifications in the
above-described methods, and compositions can be made
without departing from the spirit and scope of the
invention. Accordingly, the invention may be embodied
in other specific forms without departing from the
spirit or essential characteristics thereof. Present
examples and embodiments, therefore, are to be
considered in all respects as illustrative and not
restrictive, and all changes which come within the
meaning and range of equivalency of the specifications
are therefore intended to be embraced therein.
