QUANTITATIVE CYTOFLUOROMETRIC 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-lo cated SV40 large tumor antigen (large T) molecules of SV40-transformed cell lines by cytofluorimetric analysis. Five different SV40-transfor med cell lines were labelled by either a biotin- or a fluorescein-conj ugated monoclonal antibody, PAb1605, which is specific for the large T carboxy-terminus. The conjugated-antibody fluorescence signals of the stained large T molecules of transformed cells were measured via cyto fluorimetry. Comparison of the fluorescence signals of calibrated bead s bearing a known number of fluorescein molecules to the signals of co njugated PAb1605 antibodies bound on microbeads to a defined number of IgG binding sites made it possible to determine the number of antibod y-accessible large T molecules per SV40-transformed cell. The numbers (x10(-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), respective ly. Thus, the technique described allows a precise quantitation of sur face-exposed, antibody-accessible viral antigen expression. (C) 1995 W iley-Liss, Inc.