A simple semi-automated plaque method for the detection of antibody-forming cell clones in microcultures

A simple semi-automated method for the assay of large numbers of replicate microcultures for the presence of antibody-forming cell clones is described. The supernatant medium is removed from microcultures by a single sharp flick on inverting the tray. The cultured cells are mixed with 0.05 ml of a plaque-revealing mix containing indicator erythrocytes and complement and then transferred to new flat-bottomed 96-well microculture trays, using a multichannel pipette or 96-channel replicator. The tray is centrifuged, the indicator erythrocytes and cultured cells forming an even monolayer on the bottom surface of the well. Trays are held at 37 degrees C for 1-1 1/2 h to allow plaque development. Using a dissecting microscope, the number of plaques in each well is counted, or in the case of limiting dilution analysis, each well is simply scored as positive or negative. This assay procedure provides a simple, rapid and inexpensive means of assaying large numbers of microculture trays for the detection and enumeration of antibody-forming cell clones. There is no loss in sensitivity compared with the standard hemolytic plaque assay methods. The method is particularly useful for limiting dilution analysis which necessitates the assay of large numbers of replicate cultures for either the presence of absence of a clone of antibody-forming cells.