Characterization of a novel monoclonal antibody against human perforin using transfected cell lines

The role of perforin in cytotoxicity is controversial. This paper characterizes a novel monoclonal antibody (anti-Phu) against human perforin, using murine cell lines transfected with human perforin cDNA. The antibody specifically stains human perforin in transfected mouse CTLL-2. Anti-Phu blocked granule-mediated haemolysis in an in vitro assay using intact granules isolated from the natural killer (NK)-like human cell line YT, indicating that perforin is a major granule component causing lysis of red blood cells (RBC) in this assay. Inhibition of haemolysis by anti-Phu demonstrated that the antibody binds to undenatured protein as well as fixed perforin molecules. However, the antibody did not inhibit lysis by an allospecific T-cell clone or by YT cells. This could be due to an extremely tight contact between effector and target cell, preventing the antibody from interfering with perforin function by steric hindrance. Physiologically this may reduce lysis of bystander cells. The anti-Phu antibody is a useful tool for further studies of perforin-induced cytotoxicity in vitro and in vivo.