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Murine monoclonal glutamic acid decarboxylase (GAD)65 antibodies recognize autoimmune-associated GAD epitope regions targeted in patients with type 1 diabetes mellitus and Stiff-man syndrome

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Abstract

To study the immune response to glutamic acid decarboxylase (GAD) in insulin-dependent diabetes mellitus, monoclonal GAD antibodies after fusion of splenocytes from a nondiabetes-susceptible BALB/c mouse immunized with human recombinant GAD65 were generated. Of the 44 monoclonals, 35 are specific for the GAD65 isoform, whereas 9 also react with GAD67. Some 37 monoclonals, including all GAD65/67 reactive antibodies, react with GAD by Western blot analysis. The remaining 7 GAD65 monoclonals bind GAD only in an immunoprecipitation assay, which implies that they target epitopes dependent on the conformation of the GAD molecule. The125I-GAD binding of the GAD65 monoclonals reactive on Western blotting was significantly diminished by all 3 sera from Stiff-man syndrome patients but only by 3/30 (10%) sera from type 1 diabetic patients. In contrast, the 7 monoclonal antibodies reactive with a conformation-dependent GAD epitope were competitive with 83% of GAD-autoantibody-positive sera from these diabetic patients. Using chimeric GAD65/67 proteins, the epitope region targeted by these monoclonals was mapped to the middle of GAD65 (amino acids 221–442). This central conformation-dependent GAD region was also targeted by sera from patients with type 1 diabetes. In conclusion, our data show that evne after common immunization of a nondiabetes-susceptible mouse strain, monoclonals were obtained which preferentially react with the GAD65 linear amino-terminus (amino acids 4–17) and a conformation-dependent region located in the middle of GAD targeted by autoantibodies, indicating that this GAD region is not restricted to the autoimmune response associated with the Stiff-man syndrome and the bete-cell destruction in type 1 diabetes mellitus.

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Authors and Affiliations

  1. Institute of Diabetes ‘Gerhardt Katsch’ Karlsburg, Greifswalder Strasse 11a, D-17495, Karlsburg, Germany

    B. Ziegler, M. Schlosser, F. Lühder, M. Strebelow, P. Augstein & M. Ziegler

  2. ELIAS Entwicklungslabor, Freiburg, Germany

    W. Northemann

  3. Division of Endocrinology, Vanderbilt University, Department of Veteran Affairs, Medical Center, Nashville, Tennessee, USA

    A. C. Powers

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  1. B. Ziegler
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  2. M. Schlosser
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  3. F. Lühder
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  4. M. Strebelow
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  5. P. Augstein
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  6. W. Northemann
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  7. A. C. Powers
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  8. M. Ziegler
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Ziegler, B., Schlosser, M., Lühder, F. et al. Murine monoclonal glutamic acid decarboxylase (GAD)65 antibodies recognize autoimmune-associated GAD epitope regions targeted in patients with type 1 diabetes mellitus and Stiff-man syndrome. Acta Diabetol 33, 225–231 (1996). https://doi.org/10.1007/BF02048548

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  • DOI: https://doi.org/10.1007/BF02048548

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