Studies on mouse Moloney virus induced tumours: I. The detection of p30 as a cytotoxic target on murine Moloney leukaemic spleen cells, and on an in vitro Moloney sarcoma line by antibody mediated cytotoxicity

Abstract

Antigenic determinants of p30, the most abundant internal virion protein of C type RNA viruses, were detected on the surface of spleen cells from mice bearing Moloney leukaemia and on an in vitro line of Moloney sarcoma, MSC. On both cell types, these determinants on the p30 molecules served as cytotoxic targets in a xenogenic complement dependent antibody mediated 51Cr release assay. Two antisera were used: a rat anti MLV -M induced lymphoma serum, and an antiserum raised in goats to either disrupted FeLV. The cytotoxic target antigens of these antisera were analysed by inhibition of cytotoxicity with viral and cellular proteins.

Studies on mouse Moloney virus induced tumours: II. Detection of p30 in the serum of mice with Moloney leukaemia by in vitro blocking of complement dependent antibody mediated cytotoxicity

“…We have not studied this antiserum with MSC targets. Since our previous study (Epstein and Knight, 1975) (Epstein and Knight, 1975) indicated that this antiserum could detect antigenic determinants of p30, the most abundant internal virion protein of the C type RNA viruses on the surface of both of these targets. We found that as little as 0418 ,/g of a purified preparation of p30 obtained by isoelectric focusing could block 4300 of the cytotoxicity of this antiserum for MSC targets and 10000 of its cytotoxicity for M targets.…”

“…Details concerning the source and maintenance of Balb/c mice, passage of Moloney leukaemia in vivo and MSC cells in vitro, preparation of antisera and rabbit complement and performance of the 5'Cr release antibody mediated cytotoxicity test are described in the previous paper (Epstein and Knight, 1975 Absorption of antisera with mouse sera.-Various volumes of mouse serum (5-50 ,ul) were combined with either neat Rat ILR-3 or goat anti-gs3 and sufficient medium (Dulbecco's modification of Eagle's medium with glutamine (E-4-G1) supplemented with 10% normal rat or goat serum) to bring the antiserum to the dilution required in a final volume of 0-1 ml. The xenogeneic serum mixtures were kept at room temperature for 30 min and then employed in the 5ICr release assay.…”

“…Antibody cytotoxicity assay.-Either M leukaemia spleen cells (5 x 105 viable) or MSC cells (1 x 105 viable) were used as targets in the assay and were labelled with 5'Cr as described previously (Epstein and Knight, 1975). For each assay total 5tCr label, spontaneous 51 Cr release and maximum release by Brij detergent (polyoxyethylene lauryl ether, Pierce Chemical Co., U.S.A.) were determined.…”

“…In addition, control tubes were prepared to determine the effect of rabbit complement (1: 3), E-4-GI with 10% normal rat or goat serum, normal or leukaemic mouse serum, and combinations of these on 5'Cr release. The formulae for calculating 00 specific cytotoxicity and 0% block are ex5r-14 plained in detail in the previous paper (Epstein and Knight, 1975 The formula for calculating %0 block is described in Materials and Methods in the previous paper (Epstein and Knight, 1975 Two representative experiments are depicted in Fig. 4.…”

“…In a previous paper we demonstrated that antigenic determinants of the most abundant internal virion protein of the C type RNA viruses, p30, are present on the surface of spleen cells from mice bearing Moloney leukaemia and on an in vitro cell line derived from a Moloney sarcoma virus (MSV-M) induced tumour (MSC) (Epstein and Knight, 1975). Complement dependent antibody mediated cytotoxicity was Lused as the technique for detection of the antigenic determinants of p30, using an antiserum (ILR-3) prepared in rats against a syngeneic Moloney virus (MLV-M)-induced lymphoma and a goat antiserum (goat anti-gs3) prepared against disrupted feline leukaemia virus (FeLV).…”