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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

Department of Biochemistry, University of Ulm, P.O. Box 4066, D-7900 Ulm/Donau, Federal Republic of Germany

Simian virus 40 (SV40)-transformed cells express the SV40-specific tumour transplantation antigen (TSTA) on the cell surface and the SV40-coded tumour antigen in their nuclei. TSTA is defined by SV40-specific transplantation immunity, whereas T-antigen (T-Ag) can be detected serologically by indirect immunofluorescence. Both antigens, however, are derived from the A gene of SV40. We therefore analysed SV40-transformed cells 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. However, 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-denatured T-Ag, but not with sera from hamsters bearing SV40-induced tumours. 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 tumour 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 tumour 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-labelled Staphylococcus aureus to the cell surface. Our results are consistent with the hypothesis that SV40 T-Ag-related antigens are involved in the formation of TSTA.

Received 12 June 1980; accepted 22 September 1980.

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