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Isolation of Meth A Cell Surface Membranes Possessing Tumor-specific Transplantation Antigen Activity¹

Department of Surgery, Francis Delafield Hospital, College of Physicians and Surgeons of Columbia University, New York, New York 10032

A method is described for isolating the surface membranes from both transplanted Meth A ascites tumor cells in BALB/c mice and the leukemic cells of AKR/J mice. These cells are harvested and washed several times in a solution of 150 mM NaCl, 50 mM borate, 1.0 mM CaCl₂, and 1 mM MgCl₂, pH 7.2, at 0°. The cells are then extracted at room temperature in large volumes of 0.2 mM EDTA and either 2.5 mM borate (Meth A) or 20 mM borate (leukemic) at pH 9.2. After extraction, the further addition of 20 mM borate, pH 9.2, precipitates out most of the intracellular structures. The membranes are freed of contaminating particles by washes in dilute borate. The final product consists essentially of empty bags, the surface membrane ghosts. Chemical and electron microscopic studies confirm the almost exclusive recovery of surface membranes. The Meth A membranes possess tumor-specific transplantation antigens according to the transplantation challenge test in BALB/c mice, the strain of origin of the Meth A tumor. The leukemic cell membranes do not immunize against Meth A tumor. The onset of detectable immunity is quite rapid, reminiscent of second set rejection of skin grafts following i.p. sensitization with donor spleen cells. Isolated Meth A surface membranes will be used for immunogenicity studies of tumor transplantation antigens.

¹ Supported in part by American Cancer Society Grant T-446 and Damon Runyon Memorial Fund for Cancer Research Grant DRG-1023.

Received 3/12/70. Accepted 7/31/70.