Because of their differential expression on T-cell subsets, Lyt antigens are important markers for studies of T-cell differentiation (Cantor and Boyse 1976). The first monoclonal antibodies produced against Lyt antigens were xenogeneic (rat anti-Lyt-1 and anti-Lyt-2; see Ledbetter and Herzenberg 1979). These antibodies did not distinguish between allelic forms of the Lyt-1 and Lyt-2 antigen and were not complement-binding. Later, complement-fixing monoclonal mouse antibodies against the Lyt-l.1, Lyt-2.1, and Lyt-2.2 antigens were produced (Hogarth et al. 1980, U. H~immerling, personal communication). However, monoclonal antibody against the Lyt-l.2 antigen (the more frequent of the two alMic forms of the Lyt-1 antigen) has only been available commercially. Here we describe a strongly cytotoxic monoclonal Lyt-l.2-specific antibody produced by a hybridoma line that is available to interested investigators.
The hybridoma line, which we designate C3PO, was produced thus: mice of the C3H/HeJ strain received four intraperitoneal injections of CE/J thymocytes (approximately 5 x 107 cells per injection) at bi-weekly intervals, and then one intravenous injection of 5 x 106 thymocytes 3 days before fusion. Spleen cells were hybridized with the nonsecretor SP2/O-Agl4 myeloma line according to the protocol described by Lemke and his co-workers (1978) with slight modifications, using 50% polyethylene glycol, M r 1000 (Sigma, Miinchen, Federal Republic of Germany). The fused cells were cultured in selective HAT medium at a density of approximately 2 x 105 cells per well in 96-well tissue culture plates (Dynatech, Niirtingen, Federal Republic of Germany). Supernatants from wells containing growing hybrids were tested against the immunizing cells in the two-stage microcytotoxicity assay (Klein et al. 1975). Eight of 808 growing hybrids were positive, and one of them was established as a cell line. The line was then cloned by limiting dilution, yielding ten positive clones. The antibodies produced by the clones (either as tissue culture supernatants or as ascites fluid) were panel tested.
The antibody reacts with cells of Lyt-l.2-positive strains such as A/J, BALB/c C57BL/6, 129/St, and CE, but does not react with cells of Lyt-1.2-negative (Lyt-1.