[Detection and isolation of antibody-forming clones by means of local cytolysis in gel]

Abstract

A method is suggested combining the screening and cloning of hybridomas producing cytotoxic antibodies. The method is based on the Erne local hemolysis principles. The cells from the preformed hybridoma line NATF 9.9 secreted monoclonal antibodies (McAb) against murine T lymphocyte differentiation antigen Lyt-3.2. A mixture of hybridoma cells and target cells was attached to the plastic Petri dish surface pretreated with Poly-L-lysine and covered with agarose. McAb production by hybridoma cells was elicited by complement-dependent lysis of target cells. The lysis was detected by incorporation in dead cells of the fluorescent dye ethidium bromide. Subsequent studies made it possible to evaluate the clonogenic capacity of McAb production and isolate active clones.