Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2000-10-17
2004-03-09
Le, Long V. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007400, C435S007500, C435S007920, C435S021000, C435S040500, C436S063000, C436S086000, C436S151000, C424S130100, C424S134100
Reexamination Certificate
active
06703208
ABSTRACT:
BACKGROUND OF THE INVENTION
1. 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.
2. 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 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 Ca
2+
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 &agr;-carboxyl groups of glutamine residues in one polypeptide and &egr;-NH
2
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 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 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 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 250 ng/ml to 3 &mgr;g/ml. Next, the solid matrix can be coated with tTG. In one embodiment, the concentration of tTG for coating was 5 ug/ml to 20 ug/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 bind
Kumar Vijay
Rajadhyaksha Manoj
Cheu Changhwa J
Hodgson & Russ LLP
Immco Diagnostics
Le Long V.
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