Production of monoclonal antibodies to islet cell surface antigens using hybridization of spleen lymphocytes from non-obese diabetic mice

Abstract

Non-obese diabetic mice display a syndrome with dramatic clinical and pathological features similar to those of Type 1 (insulin-dependent) diabetes in man. Circulating autoantibodies to the surface of islet cells were demonstrated in some of these mice by a protein A radioligand assay. To produce monoclonal antibodies to islet cell surface antigens, therefore, we took the spleens of non-obese diabetic mice, transferred the spleen cells into non-immunized recipient mice, which were made immunologically incompetent by a large dose of X-irradiation, and then fused their lymphocytes with FO mouse myeloma cells. After screening the resultant hybrids, one stable hybridoma (3A4) that produced a monoclonal antibody (IgG1) specifically bound to the surface of islet cells was obtained. The purified monoclonal antibody was bound to the surface of transplantable Syrian golden hamster insulinoma cells sevenfold more than control antibody. Adsorption of the antibody on mouse spleen lymphocytes or thymocytes resulted in only a slight decrease in 125I-protein A binding to insulinoma cells. This antibody also reacted with the surface of mouse and rat islet cells, but not with that of rat spleen cells or hepatocytes. A spectrophotometric assay for peroxidase activity demonstrated that six times more peroxidase bound to insulinoma cells incubated with the antibody than to cells treated with control antibody. Furthermore, this antibody could be visually detected in the immunoenzymatic labelling of the surface of insulinoma cells. In summary, we have developed a novel method of producing monoclonal antibodies to the surface of islet cells for probing into the pathogenesis of Type 1 diabetes.

Monoclonal antibody-mediated cytotoxicity against rat Beta cells detected in vitro does not cause Beta-cell destruction in vivo

“…Independent of the different regimes used to establish primary induction the two monoclonals, M10H6 and K14D10 showed striking surface binding to both the pri-mary rat islet cells and the insulin-producing RIN cells, which confirms similar results observed with other mc-ICSA [13,[22][23][24][25]. The insulinoma cells obviously share many antigens of normal pancreatic islet cells and are suitable for detecting not only mc-ICSA but also polyclonal ICSA in Type i diabetic sera [9,26,27].…”

“…Interestingly, both surface antibodies react as ICA on rat pancreatic cryosections, which also means the monoclonal antibodies K14D10 and M10H6 recognize antigens which occur in the cytoplasm as well as on the surface of Beta cells. This dual reactivity as ICSA and ICA has been described previously by Eisenbarth et al [28] for the mc-ICSA A2Bs, whereas most mc-ICSA failed to label the islets of pancreatic cryostat sections [23,24]. Probably cell surface antigens are less abundant on pancreatic cryostat sections.…”

Dependence of Antigen Expression on Functional State of β-Cells

“…Studies to determine the corresponding antigen are in progress. Other MoAbs have been produced from the BB rat (22) and the NOD mouse (7), but none has obtained the same degree of specificity as IC2. In this study, we substantiated that, among islet cells, IC2 specifically binds to p-cells and does not bind to insulin.…”

Experimental Models of Type-I Diabetes

“…Note however that anti-insulin anti bodies may be detected earlier [Michel, in prep.]. Note also that ICSA (detected by a RIA) have been obtained in the monoclonal form after transfer of NOD mouse spleen cells in irradiated recipients [124] or more simply by direct fusion [Boitard, in prep. ].…”

“…However, anti-islet surface autoantibodies may be found by 5lCr release [ 123], radioimmunoassay (RIA) [ 124] or inhibition of insulin release by rat islets in vitro [ 123], Such antibodies are detected late in the life of NOD mice and mainly in females at the time of onset of overt dia betes. Note however that anti-insulin anti bodies may be detected earlier [Michel, in prep.].…”