A flow cytometric micromethod for the detection of Fcϵ receptors and IgE binding factors using fluorescent microspheres

Abstract

Two assays based on the use of fluorescent microspheres have been developed in order to detect Fcϵ receptors on human cells and human IgE binding factors. A direct assay using microspheres to which IgE was coupled permitted the detection of Fcϵ receptors on RPMI 8866 cells. However the fluorescence intensity was relatively low and made it difficult to discriminate between positive and negative cells. To increase the sensitivity of the assay, an indirect 3-step test was developed, in which the cells were subsequently incubated with soluble IgE, a polyclonal or monoclonal anti-IgE antibody and fluorescent microspheres to which anti-mouse or anti-rabbit immunoglobulin was coupled. This indirect assay resulted in an increased fluorescence intensity. Optimal discrimination between positive and negative cells was obtained. This assay permitted the detection of human IgE binding factors produced by RPMI 8866 cells. The binding of IgE was not dependent on the origin of the latter. Among the different cell lines tested, the EBV positive lymphoblastoid cells were found to express Fcϵ receptors and to release IgE binding factors in their supernatants.