Screening and replica plating of anti-hapten hybridomas with a transfer template hemolytic spot assay

A localized hemolysis in gel assay is described for screening microcultures of hybridomas for the production of anti-hapten antibody. The keys to the rapid screening assay reported here are a special fenestrated transfer template and an improved hapten conjugated target cell. The transfer template is a 96-well plate with a calibrated hole in the bottom center of each well. To assay for anti-hapten antibodies, the transfer template is positioned over a 96-well microculture plate containing the growing hybridomas. After making contact with the tissue culture supernatant each orifice of the transfer template retains approximately 2 microliter of tissue culture supernatant. The transfer template is then placed onto an assay slide containing a thin layer of hapten conjugated target erythrocytes incorporated into agarose. After incubation with an anti-immunoglobulin and complement, areas of localized hemolysis in the gel indicate hybridomas which are secreting anti-hapten antibodies. The assay detects as little as 10 pg of antibody. Since the transfer template can be used as a replica plate, one can repeatedly transfer samples to various slides which contain either different hapten target cells or different hapten analog inhibitors in the agarose layer. Therefore, in addition to rapidly screening microcultures for positive hybridomas this procedure permits the characterization of each monoclonal antibody's fine specificity.