A DOT-IMMUNOBINDING ASSAY FOR THE DEMONSTRATION OF SOLUBLE FC-GAMMA RECEPTORS

We have developed a sensitive dot-immunobinding assay to demonstrate and characterize the functional activity of soluble Fc gamma receptors (FcR). Samples containing soluble FcR were immobilized on a nitrocellulose membrane, Immune complexes of horseradish peroxidase and rabbit IgG antibodies to horseradish peroxidase (HRP) were allowed to react with nitrocellulose-bound FcR, and the immune complexes were visualized by HRP developer. The intensity of the grey dots reflected the amount of immune complex bound. Binding of immune complexes to placental extract containing soluble FcR was inhibited completely by IgG and Fc fragments, but not by F(ab')(2) fragments, IgA and IgM. The method was used to characterize the subclass specificity of solubilized placental FcR. Human Fc fragments, and intact IgG1 and IgG3 proteins inhibited the binding whereas preparations of F(ab')(2), IgG(2) and IgG4 did not. In conclusion, the dot-immunobinding assay described is a rapid and simple method for the demonstration and characterization of functionally active soluble FcR.