International Workshop on Lessons From Animal Models for Human Type 1 Diabetes: identification of insulin but not glutamic acid decarboxylase or IA-2 as specific autoantigens of humoral autoimmunity in nonobese diabetic mice.

Several self-antigens have been reported as targets of the autoimmune response in nonobese diabetic (NOD) mice. The aim of this workshop was to identify autoantibody assays that could provide useful markers of autoimmunity in this animal model for type 1 diabetes. More than 400 serum samples from NOD (4, 8, and 12 weeks of age and at diabetes onset), BALB/c, and B6 mice were collected from six separate animal facilities, coded, and distributed to five laboratories for autoantibody measurement. Insulin autoantibodies (IAA) were measured by radiobinding assay (RBA) by four laboratories and by enzyme-linked immunosorbent assay (ELISA) in one laboratory. Using the 99th percentile of BALB/c and B6 control mice as the threshold definition of positivity, IAA by RBA were detected in NOD mice at frequencies ranging from 10 to 30% at age 4 weeks, from 26 to 56% at 8 weeks, from 42 to 56% at 12 weeks, and from 15 to 75% at diabetes onset. With ELISA, IAA signals differed significantly between control mouse strains and increased with age in both control and NOD mice, with frequencies in NOD animals being 0% at 4 weeks, 14% at 8 weeks, 19% at 12 weeks, and 42% at diabetes onset. For IAA, the ELISA results were relatively discordant with those of RBA. GAD autoantibody (GADA) and IA-2 autoantibody (IA-2A) signals obtained by RBA were low (maximum 2.5% of total) but were increased in NOD mice compared with control mice at diabetes onset (GADA 29-50%; IA-2A 36-47%). ELISA also detected GADA (42%) and IA-2A (50%) at diabetes onset, with results concordant with those of RBA. Remarkably, GADA and IA-2A frequencies varied significantly with respect to the source colony of NOD mice. Furthermore, whereas neither GADA nor IA-2A correlated with IAA, there was strong concordance between GADA and IA-2A in individual mice. Sera with increased binding to GAD and IA-2 also had increased binding to the unrelated antigen myelin oligodendrocyte glycoprotein, and binding to GAD could not be inhibited with excess unlabeled antigen, suggesting nonspecific interactions. In sum, this workshop demonstrated that IAA measured by sensitive RBA are a marker of autoimmunity in NOD mice and draw into question the true nature of GADA and IA-2A in this animal model.