Cross-Linking of IgE-Receptor complexes at the cell surface: A fluorescence method for studying the binding of monovalent and bivalent haptens to IgE

Abstract

We have developed a method for use in investigating factors controlling the binding and cross-linking by bivalent haptens of immunoglobulin E (IgE) bound to receptors on rat basophilic leukemia (RBL) cells. This method employs monoclonal anti-2,4-dinitrophenyl (DNP) IgE that is labeled with fluorescein-5-isothiocyanate (FITC), and it measures FITC quenching that accompanies DNP occupation of the antibody combining sites in a titration experiment. The validity of this approach is demonstrated using the monovalent hapten DNP- l-lysine. The affinity constant for this ligand obtained by the FITC quenching method is compared with those obtained with previously established methods: equilibrium dialysis and quenching of endogenous tryptophan for IgE in solution and [ 3H]-DNP- l-lysine binding to IgE on cells. The FITC quenching method has been used to carry out a detailed study of the binding of monovalent DNP-aminocapryol l-tyrosine (DCT) and bivalent (DCT) 2-cystine to FITC-IgE and its Fab fragments in solution. Intrinsic ( K) and cross-linking ( K x) affinity constants are obtained by analyzing the binding curves in terms of simple equilibrium equations. With these DCT haptens the ability of this method to assess hapten binding and cross-linking of IgE bound to receptors on RBL cells is shown.