Biochemical purification of tumoricidal factors obtained from human plasma

Continuous DEAE-Sephadex ion-exchange gradients were utilized to purify 2 human plasma factors (A and B) which possessed cytotoxic activity in vitro against human tumor cells but not against normal cells. Antibodies were elicited in rabbits against human factors A and B. After insolubilization of the rabbit anti-human tumoricidal factor antibodies, the 2 factors, could then be purified utilizing immunoadsorbent techniques. Human plasma was adsorbed to the insolubilized antibodies so that factors A and B could be isolated in quantities large enough to conduct biologic studies. When the 2 factors purified by DEAE ion-exchange chromatography were examined by SDS-polyacrylamide gel electrophoresis, both factors A and B consisted of 1 protein band with an approximate molecular weight of 67,000. Both factors A and B were destroyed by heating at 100 degrees C. However, when heated at 56 degrees C for 30 min, factor A was destroyed whereas factor B retained its cytotoxic effects. Furthermore, the mechanism of tumor killing by the human factors appears to be by means of a soluble mediator. The addition of arginine to the incubation mixture had no effect on cytotoxicity. However, incubation with catalase or superoxide dismutase suppresses cytotoxicity. Thus it appears that O2- or H2O2 functions as an anti-tumor substance in our system. When the kinetics of tumor killing were studied, it was found that human factors were non-cytotoxic after 4 hr incubation, possessed slight cytotoxicity at 8 hr but reached maximal cytotoxicity levels after 24 hr incubation.