Abstract
In the present study, human hybridoma techniques were employed to study autoreactive lymphocytes present in the peripheral blood of a patient with acute onset Type 1 (insulin-dependent) diabetes mellitus. Peripheral blood lymphocytes from this patient were fused with mouse myeloma NS1 cells. Resulting hybridomas were selected by their growth in HAT medium. Hybridomas secreting human immunoglobulins were identified by a two-site immunoradiometric assay. After cloning and expansion in tissue culture, nineteen cell lines were obtained that secreted human immunoglobulin. Two cell lines, HML3. 21 and HML3. 22 were characterized. Karyotype analysis confirmed that the cell lines were human-mouse heterohybridomas. Immunodiffusion, immunofixation, and SDS-PAGE studies showed that the two monoclonal antibodies were human IgG (k-light chain) class. Immunohistochemistry and thyroid assays indicated that HML3. 21 identified thyroglobulin, while HML3. 22 recognized an epitope on the TSH receptor. The study demonstrated the potential usefulness of human monoclonal antibodies as tools to study autoimmunity in Type 1 diabetes mellitus. Two assays were developed for the detection of islet cell surface antibodies (ICSA). Both assay systems employed the rat insulin-secreting line RINm5F. Optimization and validation studies were performed. The cell immunoradiometric assay (IRMA) could be performed with a 60 minute incubation of sample followed by a 60 minute incubation with radiolabelled second antibody. A rapid ELISA for islet cell surface antibodies was developed. Sera from Type 1 diabetic patients were incubated for 30 minutes with RINm5F cell membrane antigens pre-coated onto microtitre wells. Bound immunoglobulins were detected by a further incubation with an enzyme-labelled second antibody for 30 minutes. No significant cross-reactivity was observed in the assay for other autoantibodies. There was good correlation between the two assay systems.