Quantitative Fluorometric Assay for Detection and Characterization of Fc Receptors

In studies, the quantitative measurements of the binding parameters of heat-aggregated IgG, chemically cross-linked IgG polymers, and immune complexes to FcγR on several cell types were obtained by radioreceptor assays. In this chapter, the quantitative fluorometric assay is discussed to represent a simple alternative to these radio labeling techniques. A fluorophore moiety is chemically coupled to the appropriate ligand, and the assay is based on the quantitation of the cell-associated ligand by spectrofluorometry. In the experiment discussed in the chapter, rabbit IgG was used as ligand because its heat-aggregates readily and binds well to human, rabbit, and murine FcγR, while avoiding problems of subclass specificity. It is concluded that fluorescence values of the nonspecific binding points have to be subtracted from the values of the experimental points to obtain the specific cell-bound fluorescence. Fluorescence is measured in arbitrary units. To convert the cell-bound fluorescence into the concentration of bound aggregates, a standard plot of fluorescence intensity versus the total concentration of aggregates in the presence of cell lysates has to be experimentally established in parallel with each assay. The linearity of this plot is conserved over fluorophore concentrations ranging from 10–11 M to 10–5 M, thus, it is widely covering the range of concentrations used for the FcγR assay.