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.
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. The 125I-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 even after common immunization of a nondiabetes-susceptible mouse strain, monoclonal 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 beta-cell destruction in type 1 diabetes mellitus.
Ziegler
B
B
Institute of Diabetes Gerhardt Katsch Karlsburg, Germany.
Schlosser
M
M
Lühder
F
F
Strebelow
M
M
Augstein
P
P
Northemann
W
W
Powers
A C
AC
Ziegler
M
M
D000818:Animals; D000911:Antibodies, Monoclonal; D001323:Autoantibodies / Q000097:blood*; D001921:Brain / Q000201:enzymology; D003922:Diabetes Mellitus, Type 1 / Q000097:blood / Q000276:immunology*; D004198:Disease Susceptibility; D000939:Epitopes / Q000032:analysis* / Q000276:immunology; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect; D005968:Glutamate Decarboxylase / Q000276:immunology*; D006801:Humans; D007515:Islets of Langerhans / Q000201:enzymology / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C / Q000276:immunology; D011487:Protein Conformation; D051381:Rats; D011994:Recombinant Proteins / Q000276:immunology; D016750:Stiff-Person Syndrome / Q000097:blood / Q000276:immunology*
D000911:Antibodies, Monoclonal; D001323:Autoantibodies; D000939:Epitopes; D011994:Recombinant Proteins; D005968:Glutamate Decarboxylase