Rapid screening and selection of monoclonal antibodies by bivariate flow cytometric analyses

We describe a bivariate flow cytometric assay to rapidly identify hybridomas producing new monoclonal antibodies recognizing subpopulations that are unreactive with existing immunological reagents. In this screen, whole cells in microtiter wells are labeled first with a red-linked test antibody, and then with a green-linked cocktail of existing monoclonal antibody reagents. The multiply-stained fluorescent cells are analyzed flow cytometrically and bivariate distributions of red vs. green-linked antibody fluorescence are generated. Test antibodies that recognize different subpopulations than those labeled by antibodies in the cocktail are readily identified. The use of an antibody cocktail conjugated with a single fluorophore allows comparison of the reactivity of the test antibody with multiple existing antibodies in a single analysis. This screen allows rapid (approximately 100 test antibodies can be evaluated in 40 min) identification of potentially interesting new antibodies for discrimination of subpopulations in heterogeneous tissues. We describe application of this assay to identify antibodies useful to mark hemopoietic subpopulations.