Antigenic Differences between Normal and Polyoma Virus-transformed Cells. Ii. in Vitro Evidence for a Virus-induced Antigen.

that cells transformed by the polyoma virus possess a specific antigen which can be detected by trans plantation experiments. It would be desirable, for many reasons, to identify this antigen by an in vitro reaction. For this purpose, attempts to prepare heterologous anti sera selective for polyoma-transformed hamster and mouse cells were made. A set of experiments, already reported (1), failed because of extensive cross-reaction of the sera with malignant cells of nonpolyoma origin. Further studies wifi now be reported which have been more suc cessful. The sera obtained showed specificity, since they sensitized most strongly cells of hamster and of mouse origin transformed by polyoma virus. Even with these sera, however, the effect was not yet entirely specific. MATERIALS AND METHODS Details of methodology have been presented pre viously (1). In all assays suspensions of single cells were exposed to absorbed antiserum in the absence of comple ment for 1 hour at 37°C. They were centrifuged and then resuspended in 5 per cent unheated normal rabbit serum (as complement) in modified Eagle's medium with 10 per cent calf serum. Cell samples were withdrawn at intervals and assayed for survivors by measuring the colony-forming capacity of the cells in each sample popu lation. An antiserum's capacity to sensitize cells during the first phase was eventually expressed by the rate and extent of cell kffling by complement (5 per cent rabbit serum) in the second phase. This two-step reaction suc cessfully avoided the anti-complementary effects of materials found in absorbed sera. Complement alone killed certain cell types more or less rapidly. Therefore, as a control, an aliquot of each cell suspension was treated with the same rabbit's heat inactivated (56° C., 30 minutes) pre-immune serum. Then both aliquots were handled in exactly the same way in the complement phase. Killing rates for the two aliquots were compared. Cells exposed to heat-inactivated pre-immune sera or to absorbed sera (which had been decomplemented by ab sorption) suffered no loss of viability even after 1 hour. Killing of such sensitized cells was entirely confined to the complement phase of each experiment. Sensitizations were carried out at a cell concentration of 8 X 10@per ml. or 1.6 X 10@ per ml. For the complement phase cells were resuspended at 3 X 10@per ml. ; 600†" 6000cells were then plated for survivors in replicate 4-oz. bottles and were incubated in a 5 per cent CO2 …