We have produced a monoclonal antibody 3A4 to the surface of islet cells by fusing spleen lymphocytes from nonobese diabetic (NOD) mice. To identify the molecular weight of specific target antigens reacting with 3A4, 125I-surface-labeled In-111 insulinoma cells were solubilized and extracts were absorbed with 3A4, and immunoprecipitates were followed by polyacrylamide gel electrophoresis and autoradiography. 3A4 recognized two major polypeptides with apparent molecular weights of radioactive 64K and inactive 28K. In order to evaluate the antibody-mediated cytotoxic mechanisms of 3A4, complement-dependent antibodymediated cytotoxicity (C'-AMC) and antibody-dependent cellular cytotoxicity (ADCC) were tested using a method of specific 51Cr release. In the study for C'- AMC, even over wide ranges of concentration of antibody and rabbit complement, purified 3A4 had no apparent cytotoxic effects on ln-111 cells. On the other hand, significant ADCC was observed at an antibody concentration of 10 μg/ml and a target:effector cell ratio of 1 40 (P < 0.01). Finally, the effects of 3A4 on glucose-stimulated insulin release were examined in isolated rat islets. At a glucose concentration of 16.7 mM, 3A4 significantly inhibited the insulin release either in the presence or absence of complement (P < 0.01).
In conclusion, 3A4 could not only bind but also be active to the target cells. Therefore, this monoclonal antibody should be a useful tool to permit a detailed analysis of the pathogenesis of type I diabetes mellitus.
