AUTOANTIGENIC PROPERTIES OF NATIVE AND DENATURED GLUTAMIC-ACID DECARBOXYLASE - EVIDENCE FOR A CONFORMATIONAL EPITOPE

The frequency of antibodies to GAD (anti-GAD) in insulin-dependent dia betes mellitus (IDDM) varies greatly according to the type of assay em ployed. We therefore examined the immunoassay characteristics of diabe tic sera using GAD purified from porcine brain and shown to contain bo th isoforms. Sera from 38 patients with IDDM, including 1 patient with both stiff-man syndrome (SMS) and IDDM, were studied for anti-GAD by radioimmunoprecipitation (RIP), Western blotting, and dot-blotting. Th e sera were selected according to reactivity in the RIP assay. There w as a good correlation between potency of the RIP reaction at the scree ning dilution of 1:2 and the endpoint dilution in the assay which rang ed from 1:2 to 1:30,000 for IDDM sera, and 1:300,000 for the SMS serum . Of the 38 sera positive for anti-GAD by RIP, only 6 had antibodies d etectable by Western blotting, and all gave an RIP titer of at least 1 :250. The low frequency of antibodies by Western blotting was explicab le by denaturation of the antigen. Thus, using a dot-blotting assay in which reactivity to untreated ''native'' GAD was compared with reacti vity to GAD after denaturation by reduction with 2-mercaptoethanol and boiling, 20 of the 38 IDDM sera reacted unequivocally with the native GAD compared with only 2 that reacted with denatured GAD after reduct ion and boiling. The sera were tested for their capacity to inhibit th e catalytic activity of GAD, but only the high-titer serum from the pa tient with SMS did so. Our study further validates the RIP assay for a nti-GAD and establishes that anti-GAD exists in IDDM over a wide range of titers that correlate with other assay characteristics, and also i ndicates that the conformational autoantibody epitope on GAD is suscep tible to alteration under denaturing conditions. (C) 1994 Academic Pre ss, Inc.