Longitudinal epitope analysis of insulin-binding antibodies in type 1 diabetes

Autoantibodies to insulin (IAA) are one of the first markers of the autoimmune process leading to type 1 diabetes (T1D). While other autoantibodies in T1D have been studied extensively, relatively little is known about IAA and their binding specificities, especially after insulin treatment is initiated. We hypothesize that insulin antibodies (IA) that develop upon initiation of insulin treatment differ in their epitope specificities from IAA. We analysed insulin antibody binding specificities in longitudinal samples of T1D patients (n = 49). Samples were taken at clinical diagnosis of disease and after insulin treatment was initiated. The epitope specificities were analysed using recombinant Fab (rFab) derived from insulin-specific monoclonal antibodies AE9D6 and CG7C7. Binding of radiolabelled insulin by samples taken at onset of the disease was significantly reduced in the presence of rFab CG7C7 and AE9D6. rFab AE9D6 competed sera binding to insulin significantly better than rFab CG7C7 (P = 0.02). Binding to the AE9D6-defined epitope in the initial sample was correlated inversely with age at onset (P = 0.005). The binding to the AE9D6-defined epitope increased significantly (P < 0.0001) after 3 months of insulin treatment. Binding to the CG7C7-defined epitope did not change during the analysed period of 12 months. We conclude that epitopes recognized by insulin binding antibodies can be identified using monoclonal insulin-specific rFab as competitors. Using this approach we observed that insulin treatment is accompanied by a change in epitope specificities in the emerging IA.