Detection of Simian Virus 40 T-Antigen-related Antigens by a 125I-Protein A-binding Assay and by Immunofluorescence Microscopy on the Surface of SV40-transformed Monolayer Cells

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Abstract
SV40-transformed cells express the SV40-specific tumor transplantation antigen (TSTA) on the cell surface and the SV40-coded tumor antigen [T-Ag] in their nuclei. TSTA is defined by SV40-specific transplantation immunity, whereas T-Ag can be detected serologically by indirect immunofluorescence. Both antigens are derived from the A gene of SV40. SV40-transformed mouse fibroblast 3T3 cells were analyzed for the presence of serologically detectable T-Ag-related molecules. Such antigens could not be detected on the surface of living SV40-transformed cells in monolayers. After a short formaldehyde fixation it was possible to stain the cell surfaces of SV40-transformed cells with sera from rabbits immunized with purified SDS[sodium dodecyl sulfate]-denatured T-Ag, but not with sera from hamsters bearing SV40-induced tumors. T-Ag-related antigens could be detected with both types of antisera by applying a more sensitive 125I-protein A assay. The T-Ag specificity of the binding of hamster SV40 tumor sera was demonstrated by a 125I-IgG-blocking assay in which preincubation of formaldehyde-fixed SV40-transformed cells with rabbit anti-SDS-T-Ag serum inhibited the binding of hamster SV40 tumor serum by about 70%. The localization of T-Ag-related antigens on the outside of plasma membranes of formaldehyde-fixed cells was shown by an anti-SDS-T-Ag serum-specific binding of fluorescein isothiocyanate-labeled Staphylococcus aureus to the cell surface. Results are consistent with the hypothesis that SV40 T-Ag-related antigens are involved in the formation of TSTA.

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