Analysis of anti-gliadin antibodies by immunoblot analysis and enzyme-linked immunosorbent assay using gliadin fractions as antigens

Background: Anti-gliadin antibody (AGA) determination has been widely used in the screening test to detect celiac patients in the gene;al population a nd in risk groups. Serological assays present variable efficiency, probably caused by differences in the antigenic mixtures employed as antigen, The o bjective of this wort is to evaluate the use of purified gliadin fractions in an enzyme-linked immunosorbent assay (ELISA) test. Methods: Anti-gliadin antibody reactivity was characterized in the sera of patients with celiac disease, and AGA levels were determined by immunoblot analysis using, purified gliadin fractions after separation of wheat protei ns by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and acid-PAGE and after indirect ELISA. Seven antigenic mixtures were teste d: commercial gliadin, ethanolic wheat extract, and five fast protein liqui d chromatography-purified fractions (omega-gliadins, two mixtures of alpha- /beta- and beta-/gamma-gliadins). Immunoblot analysis after A-PAGE separati on showed that immunoglobulin (IgG)A reactivity was frequently more restric ted than that of IgG. Serum IgA in 15 of 23 patients showed intense reactiv ity against omega-gliadins. Results: In seven cases, only omega-gliadins were detected, To compare the efficiency of ELISA tests, serum samples of 28 patients with celiac disease and 31 control subjects were tested against the seven gliadin fractions. I mmunoglobulin G AGAs demonstrated similar levels against the different glia din fractions, whereas IgA AGAs showed a heterogeneous reactivity that depe nded on the fraction tested. The lowest number of false-positive and false- negative results was obtained when the omega-gliadin fraction was used. Par ameters for ELISA showed that the omega-gliadin fraction elicited the highe st assay efficiency for determinations of both IgA and IgG AGAs, A good cor relation was found between IgG and IgA anti-omega-gliadin and anti-endomysi al antibody determinations. Of the 28 biopsy-confirmed patients with celiac disease, 26 samples (23 positive and 3 negative) were found to have concor dant results among the three determinations. Conclusions: In this study, an intense and, in many cases, selective recogn ition of omega-gliadins was observed. Results suggest that a higher perform ance in AGA determination could be achieved using omega-gliadin as an antig en in indirect ELISA.