Quantitation of cytotoxic rat antibodies by post-assay labeling of mitogen-induced target blasts with [3H]thymidine.

Cytotoxic rat alloantibodies were quantitated using concanavalin-A induced blasts as target cells. [3H]thymidine incorporation by such cells was linearly related to their number. Serial dilutions of cytotoxic antisera were incubated with a small number of target cells in presence of rabbit, guinea pig or rat complement. Following a short incubation, cultures were pulsed with [3H]thymidine to estimate the number of live cells. Cytotoxicity titers were calculated according to conventional von Krogh analysis as the reciprocal of the dilution yielding 50% lysis. Such titers were virtually identical to titers obtained in assays in which the extent of cytolysis was determined by trypan blue or ethidium bromide exclusion. The assay, which is carried out in microtiter plates, is quantitative, economical, and objective. Furthermore, automatic harvesting of the cultures allows the rapid processing of large numbers of samples.