Significant progresses on the anti-tissue transglutaminase antibodies (anti-tTG) ELISA tests have been achieved since anti-tTG early development. Our goal was to study the optimization of the antigen immobilization and the antibody reaction conditions in an ELISA test for the detection of anti-tTG. Hence, three variants of an anti-tTG ELISA test were developed: tTG-Gliad-U, tTG-U and tTG-Gliad, according to the coating conditions (presence/absence of gliadin) and the stringency in the sample buffer (presence/absence of urea). To assess IgA and IgG anti-tTG frequencies we used a group of 117 CD patients, 36 under a gluten-free diet and 14 with a selective IgA deficiency, and a control group of 165 non-celiac patients. Sensitivity of IgA anti-tTG in CD patients with villous atrophy was 95.5% (tTG-Gliad-U), 65.0% (tTG-U) and 92.5% (tTG-Gliad), respectively. Sensitivity of the IgG anti-tTG assay was 79.0%, 21.0% and 79.0%, respectively. Specificities ranged between 92.9% and 100%. Significant lower IgA and IgG concentration values were observed in GFD CD patients. Sensitivity and specificity of IgG anti-tTG in CD patients with selective IgA deficiency were 64.3 and 100%, respectively. In conclusion, we have developed an ELISA test for IgA anti-tTG based on calcium and gliadin activated recombinant human transglutaminase. This test is sensitive and specific, and it is useful to ensure the compliance with diet of CD patients.