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 (FcepsilonRI), which is responsible for initiating
allergic reactions. To quantify
antigen-induced aggregation of
IgE-FcepsilonRI 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-FcepsilonRI aggregation. We demonstrate our method by using it to study the equilibrium binding of a haptenated fluorescent
protein, 2,4-dinitrophenol-coupled B-
phycoerythrin (DNP25-PE), 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-FcepsilonRI 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.