Characterization of anti-glycophorin monoclonal antibodies

This group of mouse monoclonal reagents comprised 31 different antibodies, 19 in the form of culture supernatants and 12 as ascitic fluids. The isotypes of antibodies in supernatant form were determined using a sandwich ELISA immunodot assay, before performing initial serological and physicochemical characterisation by microplate haemagglutination. Culture supernatants were titred without prior dilution, whereas ascitic fluids and purified supernatants were initially diluted 1 : 50 and 1 : 10 respectively in isotonic saline containing 1 % (w/v) BSA before titration. In order to determine their optimal reaction parameters, each antibody was titred at 22° C against selected M + N −, M + N + and M − N + cells at 3 different pH values − 5.5, 7.0 and 8.5. Those giving negative reactions were subjected to an indirect antiglobulin test and were then retested at 37° C and 4° C using optimal pH. Some antibodies were later found to require pretreatment with neuraminidase to induce haemagglutination. Each antibody was subsequently tested, at optimal pH and temperature, with the same M + N −, M + N + and M − N + cells, pretreated with the proteolytic enzymes ficin, trypsin and chymotrypsin plus the exoglycosidase, neuraminidase from Vibrio cholerae . Where necessary, more extensive pH studies or sequential enzyme pretreatment of cells were performed before the antibodies were divided into 3 groups - those with anti - M - like specificity, anti - N - like specificity or those showing no specificity for M - or N - antigens. Each group was then tested with selected variant cells and those with apparent anti-carbohydrate specificities were subjected to absorption with synthetic oligosaccharides linked to inorganic carriers (SYNSORBS).