Development of an immunocapture method for measuring IgA antibodies to tissue transglutaminase in the sera of patients with coeliac disease.
One of the most reliable sero-diagnostic tests for coeliac disease (CD) is the measurement, by ELISA, of serum IgA antibodies to tissue transglutaminase (tTG) adsorbed to the wells of microtitre plates. In spite of its reliability, however, some discrepancies exist with the results obtained by the antiendomysium histological assay (EMA) and by biopsy the accepted gold standard. Among the reasons for these differences in titres between the ELISA and the last 2 mentioned assays are the conformational changes that proteins undergo on adsorption and the importance of conformational epitopes on tTG for diagnosing CD. To address this problem, a novel procedure was developed using guinea-pig tTG (gptTG) free in solution to interact with IgA antibodies in the sera of CD patients. Any immune complexes so formed are then captured by anti-tTG antibodies preadsorbed to the wells of microtitre plates. This immunocapture method was optimized for the amount of soluble gptTG needed to interact with all the IgA's anti-tTG present in fixed dilutions of serum samples, the amount of rabbit IgG anti-gptTG used to coat the wells of microtitre plates and the order of addition of the reaction components. Comparison of the IgA titres obtained by immunocapture with those by EMA and ELISA (adsorbed tTG) on 9 highly positive and 6 weakly positive sera from clinically characterized CD patients and 5 negative sera from non-CD control subjects revealed that the IgA titres by the immunocapture procedure were well correlated with those obtained by EMA, whereas the titres on ELISA showed discrepancies with both immunocapture and EMA.
CovalAb, Lyon, France. elalaoui@covalab.com
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El Alaoui
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Gresti
D000918:Antibody Specificity / Q000276:immunology; D000936:Antigen-Antibody Complex / Q000276:immunology; D002446:Celiac Disease / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005903:Gliadin / Q000276:immunology; D006801:Humans; D015151:Immunoblotting / Q000379:methods; D007070:Immunoglobulin A / Q000276:immunology*; D007074:Immunoglobulin G / Q000276:immunology; D011994:Recombinant Proteins / Q000276:immunology; D015203:Reproducibility of Results; D013997:Time Factors; D011503:Transglutaminases / Q000276:immunology*
D000936:Antigen-Antibody Complex; D007070:Immunoglobulin A; D007074:Immunoglobulin G; D011994:Recombinant Proteins; D005903:Gliadin; D011503:Transglutaminases