Standartization of anti-tissue transglutaminase antibody detection and assessment of transglutaminase interactions with fibronectin: Comparison of the results with two commercially available essays

Abstract

SUMMARY OF THESIS*ABRANTES STANDARDIZATION OF ANTI-TISSUE TRANSGLUTAMINASE ANTIBODY DETECTION AND ASSESSMENT OF TRANSGLUTAMINASE INTERACTIONS WITH FIBRONECTIN. COMPARISON OF THE RESULTS WITH TWO COMMERCIALLY AVAILABLE ESSAYSINTRODUCTION: anti-endomysial (EMA) and anti-tissue transglutaminase (anti-tTg) are the main circulating autoantibodies in patients with celiac disease. Inasmuch as tissue transglutaminase (tTg) is the target antigen of EMA, specific assays have been developed to establish the serological diagnosis of celiac disease. However, published data are contradictory regarding their superiority over EMA. tTg is a cytoplasm enzyme, but indirect immunofluorescence reaction (IIF) indicates that EMA reacts with components of extracellular matrix, particularly with fibronectin. To date, serological tests have been standardized only with tTg. It is unknown if the addition of fibronectin to tTg substrate will improve the diagnostic accuracy. AIMS: 1) to standardize the indirect ELISA for detection of antitTg; 2) to standardize the indirect ELISA for detection of antibodies against the complex tTg/fibronectin; 3) to compare the results of anti-tTg, antifibronectin and anti-tTg/fibronectin antibody ELISAS of patients with not treated and treated celiac disease; 4) to compare the results of the in house indirect ELISA with available commercial kits (with tTg and htTg). MATERIALS AND METHODS: the casuistic was formed by the sera of 173 patients, including 49 with celiac disease without treatment and 124 controls (chronic diarrhea = 30; inflammatory bowel disease, n = 23; autoimmune hepatitis type 1 n = 30; treated celiac disease, n = 23; e healthy individuals, n = 18). EMA was detected by IIF, using sections of human umbilical cord as substrate. Indirect ELISA was carried out for detection of reactivity against tTg (from guinea pig liver), fibronectin (recombinant from human fibroblast) and the complex tTg-fibronectin. The performance of those essays was compared with that of anti-tTg and anti-htTg kits, purchased from Inova Diagnostics, Inc, USA. Statistical analysis was performed using Student t test, Fisher exact test, McNemar test and kappa correlation test when appropriate. RESULTS: Seropositivity to anti-tTg, antifibronectin and anti-tTg/fibronectin was observed in, respectively: 46.9%, 51% e 42.9% in the celiac disease without treatment group; 0%, 13% and 0% in the treated celiac disease group; 39.1%, 65.2% and 56.5% in the inflammatory bowel disease group; 20%, 50% and 20% in the autoimmune hepatitis group; 6.7%, 60% and 26.7% in the chronic diarrhea group. No patient among healthy controls had seropositivity to those aforementioned antibodies. Comparing the anti-tTg and tTg/fibronectin complex seropositivity in the celiac disease without treatment group, it was found that the addition of fibronectin to the tTg substrate did no improved the accuracy of the essay (p = 0.68; relative risk 1.15; 95% confidence interval = 0.7334-1.803). Comparing EMA titers with ELISA positivity, it was found that higher ser...