Rapid replicate reverse haemolytic plaque assay directly in microwells used for tissue culture

The reverse haemolytic plaque assay was adapted as a micromethod using microwells of flat-bottomed microtitre trays. To microwells containing the lymphocytes under test were added protein A-coupled ox erythrocytes, developing antisera directed against any class of immunoglobulin, and guinea pig complement absorbed with protein A-coupled erythrocytes. Plaques were scored by counting with a stereoscan microscope. The method is applicable both to spontaneous and mitogen-induced plaque formation and, as a further development of the technique to differentiated B cells in pokeweed mitogen-driven cultures, plaques were enumerated directly in the same wells of the microtitre tray as were employed for culture of the B cells. The method allows for several hundred plaque assays at one sitting.