Aggregation of ige-receptor complexes by multivalent antigens: quantifying crosslinking at the cell surface

We have investigated the equilibrium binding properties of a multivalent antigen (DNP15-Phycoerythrin) interacting with cell surface anti-DNP FITC labelled IgE (FITC-IgE). Using multiparameter flow cytometry we have developed an approach for determining the average number of crosslinks formed on the cell surface for a given ligand and IgE concentration. Fluorescence quenching that occurs when DNP binds to FITC-IgE enables one to calculate the fraction of IgE binding sites that are occupied while the number of Phycoerythrin bound per cell can be measured directly. We show that this allows one to directly determine the crosslinking curve for this multivalent antigen interacting with cell surface IgE. We examine the influence on the binding properties of cell density and the number of IgE per cell. We show mat the mass-action rate equations (mean-field equations) we use to describe binding corresponds well with the crosslinking data obtained from experiment.