Cytofluorometric quantification of cell-surface antigens by indirect immunofluorescence using monoclonal antibodies

A method has been developed to determine the absolute number of binding sites on lymphoid cells by flow cytometric analysis of indirect immunofluorescence data after saturation the related monoclonal antibodies (mAbs). First, the cell lines RPMI 8402, MOLT-4, CEM and HSB-2 were studied to determine the number of p67, T cell-associated antigen molecules expressed on their membrane, as judged by the binding of radiolabeled T101 (CD5) mAb. Then saturating doses of unlabeled T101 followed by fluorescent anti-mouse reagent were applied to these cell lines in each new experiment in order to build a standard curve relating the mean fluorescence intensity of these known cell populations to the mean number of cell-bound T101 mAb molecules. This internal standard curve was a straight line, and it was used to assess the absolute number of mAb molecules bound to other lymphoid cells. The technique was shown to be applicable to other mAb of IgG class even if used in unpurified form. These biological standards can be routinely used as internal references to establish the quantitative phenotype of lymphoid cells. The present method is referred to as quantitative indirect immunofluorescence assay (QIIF). It can be used with any flow cytometer equipped with a microcomputer.