Analysis of Anti‐Gliadin Antibodies by Immunoblot Analysis and Enzyme‐Linked Immunosorbent Assay Using Gliadin Fractions As Antigens

Abstract

ABSTRACTBackground:Anti‐gliadin antibody (AGA) determination has been widely used in the screening test to detect celiac patients in the general population and in risk groups. Serological assays present variable efficiency, probably caused by differences in the antigenic mixtures employed as antigen. The objective of this work 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 proteins by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and acid‐PAGE and after indirect ELISA. Seven antigenic mixtures were tested: commercial gliadin, ethanolic wheat extract, and five fast protein liquid chromatography‐purified fractions (ω‐gliadins, two mixtures of α‐/β‐ and β‐/γ‐gliadins). Immunoblot analysis after A‐PAGE separation showed that immunoglobulin (Ig)A reactivity was frequently more restricted than that of IgG. Serum IgA in 15 of 23 patients showed intense reactivity against ω‐gliadins.Results:In seven cases, only ω‐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. Immunoglobulin G AGAs demonstrated similar levels against the different gliadin fractions, whereas IgA AGAs showed a heterogeneous reactivity that depended on the fraction tested. The lowest number of false‐positive and false‐negative results was obtained when the ω‐gliadin fraction was used. Parameters for ELISA showed that the ω‐gliadin fraction elicited the highest assay efficiency for determinations of both IgA and IgG AGAs. A good correlation was found between IgG and IgA anti‐ω‐gliadin and anti‐endomysial antibody determinations. Of the 28 biopsy‐confirmed patients with celiac disease, 26 samples (23 positive and 3 negative) were found to have concordant results among the three determinations.Conclusions:In this study, an intense and, in many cases, selective recognition of ω‐gliadins was observed. Results suggest that a higher performance in AGA determination could be achieved using ω‐gliadin as an antigen in indirect ELISA.