A CELL-SURFACE ELISA FOR THE SCREENING OF MONOCLONAL-ANTIBODIES TO ANTIGENS ON VIABLE CELLS IN SUSPENSION

To simplify the screening of monoclonal antibodies to different human T cell surface molecules a live cell enzyme-linked immunosorbent assay (cell ELISA) has been established and optimized. The assay was perfor med in 96-well plates. By using living human T lymphocytes in suspensi on surface modification by fixation or insolubilization of the cells w as avoided. Several parameters influencing sensitivity and specificity were studied. About 150 ng/ml of mouse monoclonal antibodies to cell surface antigens could be detected when using 5 x 10(4) cells per well and a 1/1000 dilution of the anti-mouse IgG-alkaline phosphatase conj ugate. This sensitivity permitted the primary screening of cell specif ic antibodies from hybridoma supernatants. The same detection limit wa s obtained in flow cytometric analysis. If required, the sensitivity o f the cell ELISA could be increased using higher cell numbers and conj ugate concentration. When analysing different cell lines with selected antibodies the cell ELISA was found to be as sensitive and specific a s the fluorescence assay. The assay was applied to the screening of su pernatants from hybridomas developed against human T helper cell clone s and the detection of V beta specificities of T cell clones.