Quantitative cytofluorimetric determination of cell membrane‐associated large tumor antigen on SV40‐transformed cells

The aim of this study was to quantitate the number of cell membrane‐located SV40 large tumor antigen (large T) molecules of SV40‐transformed cell lines by cytofluorimetric analysis. Five different SV40‐transformed cell lines were labelled by either a biotin‐ or a fluorescein‐conjugated monoclonal antibody, PAb1605, which is specific for the large T carboxyterminus. The conjugated‐antibody fluorescence signals of the stained large T molecules of transformed cells were measured via cytotluorimetry. Comparison of the fluorescence signals of calibrated beads bearing a known number of fluorescein molecules to the signals of conjugated PAb1605 antibodies bound on microbeads to a defined number of IgG binding sites made it possible to determine the number of antibody‐accessible large T molecules per SV40‐transformed cell. The numbers (×10−4) found per cell were 1.0 (ELONA, hamster), 3.0 (VLM, mouse), 3.5 (mKSA, mouse), 11 (C57SV, mouse), and 5.5 (SV80, human), respectively. Thus, the technique described allows a precise quantitation of surfaceexposed, antibody‐accessible viral antigen expression. © 1995 Wiley‐Liss, Inc.