A new microsphere-based immunofluorescence assay using flow cytometry

The quantitative and qualitative capacities of flow cytometric analysis that have made it such a powerful tool in studies of cellular antigens have not previously been exploited when dealing with non-cellular antigens. A new immunofluorescence assay technique was developed, using an indirect staining procedure with monoclonal anti-kappa antibodies, to detect human free kappa light chains covalently bound to microspheres of a size suitable for flow cytometry. The strength of the fluorescent signal produced on the microspheres was related to the amount of antigen bound and the size of the beads. At the time of this work large microspheres (i.e., greater than 3 micron in diameter) suitable for this application were only available as suspensions of polysized beads. The fluorescent signal detected on labelled beads was optimized by selecting for analysis, on the basis of the forward angle laser scatter, only those beads of largest diameter. There are many potential applications for this technique - microspheres can be used for the presentation of virtually any antigen or antibody. The analytical benefits inherent in flow cytometry would be a significant advantage in the development of quantitative assays using this method.