Recombinant Fabs of Human Monoclonal Antibodies Specific to the Middle Epitope of GAD65 Inhibit Type 1 Diabetes–Specific GAD65Abs

  1. Carolyn J. Padoa12,
  2. J. Paul Banga3,
  3. Anne-Marie Madec4,
  4. Manfred Ziegler5,
  5. Michael Schlosser6,
  6. Eva Ortqvist7,
  7. Ingrid Kockum8,
  8. Jerry Palmer1,
  9. Olov Rolandsson9,
  10. Katherine A. Binder1,
  11. Jefferson Foote10,
  12. Dong Luo1 and
  13. Christiane S. Hampe1
  1. 1Department of Medicine, University of Washington, Seattle, Washington
  2. 2Department of Chemical Pathology, University of the Witwatersrand & National Health Laboratory Services, Johannesburg, South Africa
  3. 3Division of Medicine, Guy’s, Kings and St. Thomas’ School of Medicine, London, U.K
  4. 4Institut National de la Santé et de la Recherche Médicale, Laennec Faculty of Medicine, University of Lyon, Lyon, France
  5. 5Institute of Diabetes “Gerhardt Katsch,” Karlsburg, Germany
  6. 6Institute of Pathophysiology, Ernst Moritz Arndt University of Greifswald, Karlsburg, Germany
  7. 7Department of Woman and Child Health, Karolinska Institutet, Stockholm, Sweden
  8. 8Department of Molecular Medicine, Clinical Genetics, Karolinska Institutet, Stockholm, Sweden
  9. 9Department of Public Health and Clinical Medicine, Umeå University, Umeå, Sweden
  10. 10Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, Washington
  1. 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.

Footnotes

    • Accepted July 22, 2003.
    • Received May 8, 2003.
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  1. doi: 10.2337/diabetes.52.11.2689 Diabetes November 2003 vol. 52 no. 11 2689-2695
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