Purification of Fc_ε Receptors and Fc_γ Receptors

This chapter describes a procedure for elution of Fc_ε receptors and Fc_γ receptors that preserves their ligand-binding activity. This procedure allowed demonstrations that isolated solubilized Fc_ε receptor can bind IgE of different species and that isolated murine Fc_γ-receptor can bind to all murine IgG subclasses except IgG3.Furthermore, this elution procedure allows purifying of the receptors on fresh immunoadsorbents. Using this method, the rat Fc_ε receptor has been purified nearly to homogeneity and a receptor-associated nonglycosylated molecule has been identified. Repetitive affinity chromatography may be a generally applicable procedure for purifying other types of mammalian receptors, because it can also be used to isolate C3b receptor. The chapter also presents standardized immunoadsorbent binding assay to measure the ability of Fc_ε receptors and Fc_γ receptors to bind to their respective ligands. Radiolabeled active receptor can be incubated with 0.05–0.1 ml of immunoadsorbent in a 0.35–1.0 ml volume for 1.5 to 3 hr at 4° on an orbital shaker. Mixtures are then centrifuged at 4°, and the immunoadsorbent beads are washed 2 or 3 times at 4° in buffer containing 1% NP-40 and a carrier protein. The percentage of radioactivity bound to immunoadsorbent is a measure of receptor-binding ability. For Fc_ε receptor, the immunoadsorbent assay gives results compatible with two other independent procedures for measuring the ligand-binding ability of receptor.