Recombinant Fabs of Human Monoclonal Antibodies Specific to the Middle Epitope of GAD65 Inhibit Type 1 Diabetes–Specific GAD65Abs Carolyn J. Padoa 1 2 , J. Paul Banga 3 , Anne-Marie Madec 4 , Manfred Ziegler 5 , Michael Schlosser 6 , Eva Ortqvist 7 , Ingrid Kockum 8 , Jerry Palmer 1 , Olov Rolandsson 9 , Katherine A. Binder 1 , Jefferson Foote 10 , Dong Luo 1 and Christiane S. Hampe 1 1 Department of Medicine, University of Washington, Seattle, Washington 2 Department of Chemical Pathology, University of the Witwatersrand & National Health Laboratory Services, Johannesburg, South Africa 3 Division of Medicine, Guy’s, Kings and St. Thomas’ School of Medicine, London, U.K 4 Institut National de la Santé et de la Recherche Médicale, Laennec Faculty of Medicine, University of Lyon, Lyon, France 5 Institute of Diabetes “Gerhardt Katsch,” Karlsburg, Germany 6 Institute of Pathophysiology, Ernst Moritz Arndt University of Greifswald, Karlsburg, Germany 7 Department of Woman and Child Health, Karolinska Institutet, Stockholm, Sweden 8 Department of Molecular Medicine, Clinical Genetics, Karolinska Institutet, Stockholm, Sweden 9 Department of Public Health and Clinical Medicine, Umeå University, Umeå, Sweden 10 Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, Washington Address correspondence and reprint requests to Dr. Christiane S. Hampe, Department of Medicine, Box 357710, University of Washington, Seattle, WA 98195. E-mail: champe{at}u.washington.edu Abstract 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. APS-1, autoimmune polyendocrine syndrome type 1 CDR, complementarity-determining region GAD65Ab, autoantibody to the 65-kDa isoform of GAD LADA, late autoimmune diabetes in adults mAb, monoclonal antibody rFab, recombinant Fab RIA, radioimmunoassay Footnotes Accepted July 22, 2003. Received May 8, 2003. DIABETES