RADIOIMMUNOASSAYS FOR GLUTAMIC-ACID DECARBOXYLASE (GAD65) AND GAD65 AUTOANTIBODIES USING S-35 OR H-3 RECOMBINANT HUMAN LIGANDS

Autoantibodies are an important marker of human autoimmune diseases an d the development of simple, precise and reproducible immunoassays to detect autoantibodies is important to our understanding of human autoi mmunity. GAD65 autoantibodies occur frequently in insulin-dependent di abetic patients and is a useful marker for IDDM. A RIA to detect immun oreactive GAD65 has not been described. In the present study we descri be a semi-automated fluid-phase immunoassay for the rapid detection of GAD65 autoantibodies in human serum. We also developed a sensitive RI A to determine immunoreactive human GAD65 in biological fluids and in vitro cell systems. Using in vitro translated recombinant human GAD65 in a multiwell-adapted procedure, our GAD65Ab RIA combines high specif icity and sensitivity with a high capacity to analyze a large number o f samples. In this report the three critical steps in the GAD65Ab RIA, DNA preparation, in vitro translation and immunoprecipitation, have b een optimized. In our RIA, GAD65Ab were detected in 116/155 (75%) new onset Swedish IDDM children and in 1/85 (1.2%) healthy controls. In an immunoassay to detect autoantibodies against the proinsulin convertin g enzyme 2 (PC-2) no such antibodies were detected in IDDM patients. I n the GAD65 RIA the lower detection limit was 2 ng/ml (31 fmol/ml). Ou r data demonstrate that autoantigen radioligands produced by in vitro translation are useful in RIA for autoantibodies and autoantigens in s tudies of human autoimmunity.