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 performed in 96-well plates. By using living human T lymphocytes in suspension surface modification by fixation or insolubilization of the cells was 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 × 10 4 cells per well and a 1/1000 dilution of the anti-mouse IgG-alkaline phosphatase conjugate. This sensitivity permitted the primary screening of cell specific antibodies from hybridoma supernatants. The same detection limit was obtained in flow cytometric analysis. If required, the sensitivity of the cell ELISA could be increased using higher cell numbers and conjugate concentration. When analysing different cell lines with selected antibodies the cell ELISA was found to be as sensitive and specific as the fluorescence assay. The assay was applied to the screening of supernatants from hybridomas developed against human T helper cell clones and the detection of Vβ specificities of T cell clones.