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

Abstract

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 specmc for the large T carboxyterminus. The conjugated-antibody fluorescence signals of the stained large T molecules of transformed cells were measured via cytofluorimetry. 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 detbed number of IgG binding sites made it possible to determine the number of antibody-accessible large T molecules per SV40-transformed cell. The numbers ( X lo-*) found per cell were 1.0 (ELONA, hamster), 3.0 (VLM, mouse), 3.5 (mKSA, mouse), 11 (C57SV, mouse), and 5.5 (SVSO, human), respectively. Thus, the technique described allows a precise quantitation of surfaceexposed, antibody-accessible viral antigen expresdoll. 0 1995 WileyLiss, Inc.

Molecular quantification of cell cycle-related gene expression at the protein level

“…For the intracellular estrogen receptor, variability was higher (r ϭ 0.63) (25). Several studies have utilized this approach for surface antigens (33)(34)(35) and intracellular antigens (36) without verifying the assumptions. Given the uncertainties in this antibody-bead technology and the difficulty of independent verification for proteins that do not bind radiolabeled ligands, the data for these studies are less easily accepted in terms of molecular quantification than they are in terms of standardization.…”

Embryonic vasculogenesis by endothelial precursor cells derived from lung mesenchyme

“…Variable expression of SV40 large T antigen in transgenic mouse tumor models (Wikenheiser et al, 1992) and transformed cell lines (Hess et al, 1995) has been reported. To test the reliability of large T antigen expression, MFLM-91U were used to form tumors following subcutaneous injection in nude mice.…”