Monoclonal antibodies specific to the glutamic acid decarboxylase 65 kDa isoform derived from a non-obese diabetic (NOD) mouse.

Abstract

Two monoclonal antibodies specifically recognizing the 65 kDa isoform of the enzyme glutamic acid decarboxylase (GAD) were generated by fusion of spleen cells of a non-obese diabetic (NOD) mouse which had received a single intraperitoneal injection of 0.2 ml complete Freund's adjuvant followed three days later by one administration of a subdiabetogenic dose of streptozotocin (80 mg/kg body weight) three days before the fusion experiment was performed. Both monoclonals belong to the IgG1 isotype and were screened with an enzyme-linked immunosorbent assay using rat brain extract as a natural source of GAD and additionally with a capture assay by means of immunoglobulins of a patient with Stiff-man syndrome. The specific binding to the 65 kDa isoform of the enzyme was detected by a radioligand and an enzyme-linked immunosorbent assay using recombinant human glutamic acid decarboxylase specific for both the 67 and 65 kDa isoforms. Both monoclonal antibodies recognize the same antigenic epitope, which is located in the N-terminal region of the first 17 amino acids detected by fragments of human pancreatic 65 kDa GAD. Three out of 30 sera from Type 1 diabetic patients specifically displaced the binding of the monoclonals from 125I-labelled GAD65 measured by radio-immunoassay. A striking binding of both monoclonals M61/8F9 and M61/7E11 to the islets of cryosections of human, monkey, pig and rat pancreas but not to mouse pancreas was detectable. The antibodies failed to bind on the cell surface of viable rat islet cells. It is concluded that also in the diabetes-prone NOD mice GAD65 autoantibodies occur although GAD65 was not detectable in the mouse islets.