Different multiple reactivity of monoclonal islet cell binding antibodies using indirect immunofluorescence technique on viable cells or cellular ELISA on desiccated cells as target
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1Department of Immunochemistry, Institute of Diabetes Gerhardt Katsch, University of Griefswald, Karlsburg, Germany.
Two commonly used methods for screening hybridoma supernatants secreting monoclonal islet cell reactive antibodies (mc-ICRA) were performed to investigate the specificity of the monoclonals established. For this, endothelial, neuroblastoma, murine subcutis and two myeloma cell lines were used as targets in comparison to the insulin-producing rat insulinoma cell line (RIN), either immobilized and permeabilized in cellular enzyme linked immunosorbent assay (CELISA) or in suspension of viable cells in the indirect immunofluorescence test. In addition, rat splenocytes were used for estimating multireactivity of mc-ICRA in ELISA. Using permeabilized target cells, we obtained a high multireactivity of the monoclonal antibodies (mab) tested, indicating a high incidence of molecular mimicry between cytoplasmic antigens of different cell lines. In contrast to CELISA, if only cell surface antigens of viable cells are accessible, detected by the immunofluorescence technique, the high multireactivity is not observed. For investigating the specificity of monoclonals, the complexity of target antigens used must be taken into consideration.
Two commonly used methods for screening hybridoma supernatants secreting monoclonal islet cell reactive antibodies (mc-ICRA) were performed to investigate the specificity of the monoclonals established. For this, endothelial, neuroblastoma, murine subcutis and two myeloma cell lines were used as targets in comparison to the insulin-producing rat insulinoma cell line (RIN), either immobilized and permeabilized in cellular enzyme linked immunosorbent assay (CELISA) or in suspension of viable cells in the indirect immunofluorescence test. In addition, rat splenocytes were used for estimating multireactivity of mc-ICRA in ELISA. Using permeabilized target cells, we obtained a high multireactivity of the monoclonal antibodies (mab) tested, indicating a high incidence of molecular mimicry between cytoplasmic antigens of different cell lines. In contrast to CELISA, if only cell surface antigens of viable cells are accessible, detected by the immunofluorescence technique, the high multireactivity is not observed. For investigating the specificity of monoclonals, the complexity of target antigens used must be taken into consideration.