Development of diagnostic RI test method for antiglutamic acid decarboxylase (GAD) in SMS and IDDM patients

Western blotting with antigens purified using its specific antibody bound column has demonstrated that patients with Stiff-man syndrome (SMS) and insulin-dependent diabetic mellitus (IDDM) were both positive for anti-GAD antibody. Further, anti-GAD antibodies from various animal brains were characterized using GAD 65 and GAD 67 peptide antibody. The antibody against the anti-N-terminal peptide inhibited the enzyme activity of GAD, suggesting that the active site of GAD might exist in the N-terminal region. Development of a new detection method for anti-GAD antibody was attempted and the amount of GAD protein bound to protein G resin was determined based on the activity to release 14CO2 from 14C glutamic acid. In addition, solid-phase RIA method was developed using GAD purified by the anti-peptide antibody affinity column. The positive detection rate for GAD antibody was 39% for the enzymatic method and 56% for the solid-phase RIA method. To develop a further sensitive detection method for GAD antibody, construction of recombinant GAD was attempted and two GAD65s different in molecular size were constructed using pMal-c vector. Thus obtained antibodies against anti-N-terminal peptides were separately responded to GAD65 and GAD67 isoforms in the rat, mouse and bovine brains, whereas the carboxy-terminal antibodies were reactive to both isoforms together. Therefore, it became possible to make purification of GAD65 and GAD67 by the use of the two N-terminal peptide antibodies. Further, it became possible to purify GAD as a mixture of both isoforms. However, the yield of purification using anti-affinity column was still unsatisfactory ( several percent) and the GAD preparation obtained had little activity. The positive detection by the solid-phase RIA method was 50% for SMS patients and 56% for IDDM ones, indicating that this method was superior to the previous enzyme method. The protein A method in which labeled human recombinant GAD65 was used to precipitate 125-I GAD IgG in a test serum was significantly superior to the former two in the respects of sensitivity and specificity of the assay. (M.N.)