Detection of antibodies to islet cell and splenic lymphocytes in diabetes-prone BB and adjuvant-streptozotocin treated Lewis rats by ELISA and immunoblot analysis.

Abstract

An enzyme-linked immunosorbent assay (ELISA) has been developed to detect antibodies against surface components of rat islet and spleen lymphocytes. Live islet tumor RIN5 AH cells expressing characteristic ganglioside target antigens or rat spleen cells were immobilized onto wells of microtiter polystyrene plates precoated with poly-l-lysine and then incubated with test or normal rat sera. Cell surface-bound antibodies were quantitated after reaction with horseradish peroxidase-conjugated rabbit anti-rat Ig. With this assay, 46% (6/13) of sera from diabetes-prone BB rats and 100% (8/8) of sera from rats treated with complete Freund's adjuvant/streptozotocin (CFA/STZ) prior to immunization with RIN cells had islet cell surface antibodies: 54% (7/13) and 75% (6/8), respectively, were positive for lymphocyte antibodies (defined as the HRP anti-rat Ig binding exceeding the mean + 2SD of control group values). SDS polyacrylamide gel electrophoresis followed by immunoblotting analysis suggested that the islet cell antibodies in sera from the BB and CFA/STZ rats recognized RIN-cell components that were different in their molecular weights. These antigens were not detectable on spleen cells indicating that the ELISA described can be used to quantitate levels of islet cell specific antibodies which possibly reflect beta cell damage with progression to islet degeneration in the rat.