Detection of GAD65 Antibodies in Diabetes and Other Autoimmune Diseases Using a Simple Radioligand Assay

Abstract

Autoantibodies to glutamic acid decarboxylase (GAD) are frequent at or before the onset of insulin-dependent diabetes mellitus (IDDM). We have developed a simple, reproducible, and quantitative immunoprecipitation radioligand assay using as antigen in vitro transcribed and translated [35S]methionine-labeled human islet GAD65. By using this assay, 77% (77 of 100) of serum samples from recent-onset IDDM patients were positive for GAD65 antibodies compared with 4% (4 of 100) of serum samples from healthy control subjects. In competition analysis with unlabeled purified recombinant human islet GAD65, binding to tracer was inhibited in 74% (74 of 100) of the GAD65-positive IDDM serum samples compared with 2% of the control samples. The levels of GAD antibodies expressed as an index value relative to a standard serum, analyzed with or without competition, were almost identical (r = 0.991). The intra- and interassay variations of a positive control serum sample were 2.9 and 7.6%, respectively (n = 4). The frequency of GAD antibodies was significantly higher with IDDM onset before the age of 30 (80%, 59 of 74) than after the age of 30 (48%, 10 of 21) (P < 0.01). The prevalence of islet cell antibodies showed a similar pattern relative to age at onset. Because simultaneous occurrences of multiple autoimmune phenomena are common, we analyzed sera from patients with other autoimmune diseases. The frequency of GAD antibodies in sera positive for DNA autoantibodies (8% [2 of 25] and 4% [1 of 25] in competition analysis) or rheuma factor autoantibodies [12% (4 of 35) and 3% (1 of 35) in competition analysis] was not different from that in control samples. In contrast, in sera positive for ribonucleoprotein antibodies the frequency of GAD antibodies was significantly increased (73% [51 of 70] and 10% [7 of 70] in competition analysis [P < 0.025]). In conclusion, even large numbers of serum samples can now be tested for GAD65 antibodies in a relatively short time, allowing screening of individuals without a family history of IDDM for the presence of this marker.