Epitope analysis of GAD65Ab using fusion proteins and rFab.

The identification of disease-specific autoantibodies to the 65-kDa isoform of glutamate decarboxylase (GAD65Ab) epitopes in type 1 diabetes has been hampered by their conformational nature. Here, we compared two methods of GAD65Ab epitope analysis: GAD65/67 fusion proteins and competition assays using GAD65-specific recombinant fraction antigen binding (rFab). Sera from newly diagnosed type 1 diabetes patients (n=61) were studied using both approaches. Competition of GAD65 binding by an rFab to a specific epitope did not correlate with binding to the fusion protein that represented this epitope. Conversely, samples that bound to specific fusion proteins were not necessarily competed with rFab specific to determinants in the same region. We conclude that epitopes of different characteristics are detected by fusion proteins and by competition with rFab. Fusion proteins allow the definition of large epitope regions; however, some conformational GAD65Ab epitopes, especially those residing in the middle region, are destroyed or distorted in the fusion proteins. Competition studies using rFab allow the identification of conformational epitopes. However, monoclonal rFab may only reflect a limited proportion of the epitopes recognized by polyclonal sera. A combined analysis using both approaches may therefore be necessary to gain best understanding of autoantibody characteristics and affinity maturation.

Recombinant Fabs of human monoclonal antibodies specific to the middle epitope of GAD65 inhibit type 1 diabetes-specific GAD65Abs.

Autoantibodies to the 65-kDa isoform of GAD (GAD65Abs) are associated with type 1 diabetes development, but the conformational nature of the GAD65Ab epitopes complicates the evaluation of disease risk. Six GAD65-specific recombinant Fabs (rFabs) were cloned from monoclonal antibodies b96.11, DP-C, DP-A, DPD, 144, and 221-442. The binding of GAD65Abs in 61 type 1 diabetic patients to GAD65 was analyzed by competitive radioimmunoassays with the six rFabs to ascertain disease-specific GAD65Ab binding specificities. The median binding was reduced significantly by rFab b96.11 (72%) (P < 0.0001), DP-A (84%) (P < 0.0001), DP-C (84%) (P < 0.0001), 221-442 (79%) (P < 0.0001), and DP-D (80%) (P < 0.0001). The competition pattern in type 1 diabetic patients differed from that in GAD65Ab-positive late autoimmune diabetes in adults (LADA) patients (n = 44), first-degree relatives (n = 38), and healthy individuals (n = 14). Whereas 87 and 72% of the type 1 diabetic sera were competed by rFab b96.11 and DP-C, respectively, only 34 and 26% of LADA patients, 18 and 25% of first-degree relatives, and 7 and 28% of healthy individuals showed competition (P < 0.0001). These findings support the view that type 1 diabetes is associated with disease- and epitope-specific GAD65Abs and supports the notion that the middle epitope is disease associated. These GAD65-specific rFabs should prove useful in predicting type 1 diabetes and in the study of conformational GAD65Ab epitopes.