Quantifying aggregation of IgE-FcεRI by multivalent antigen

Aggregation of cell surface receptors by multivalent ligand can trigger a variety of cellular responses. A well-studied receptor that responds to aggregation is the high affinity receptor for IgE (FcεRI), which is responsible for initiating allergic reactions. To quantify antigen-induced aggregation of IgE-FcεRI complexes, we have developed a method based on multiparameter flow cytometry to monitor both occupancy of surface IgE combining sites and association of antigen with the cell surface. The number of bound IgE combining sites in excess of the number of bound antigens, the number of bridges between receptors, provides a quantitative measure of IgE- FcεRI aggregation. We demonstrate our method by using it to study the equilibrium binding of a haptenated fluorescent protein, 2,4-dinitrophenol- coupled B-phycoerythrin (DNP₂₅PE), to fluorescein isothiocyanate-labeled anti-DNP IgE on the surface of rat basophilic leukemia cells. The results, which we analyze with the aid of a mathematical model, indicate how IgE- FcεRI aggregation depends on the total concentrations of DNP₂₅-PE and surface IgE. As expected, we find that maximal aggregation occurs at an optimal antigen concentration. We also find that aggregation varies qualitatively with the total concentration of surface IgE as predicted by an earlier theoretical analysis.