Fluorescence protection immunoassay: a new homogeneous assay technique.
We describe a "fluorescence protection immunoassay," in which formation of an immune complex of a fluorescer-labeled antigen sterically protects the fluorescer from binding by antibodies to it. Competitive binding of unlabeled antigen by its antibody prevents formation of the fluorescer-labeled antigen immune complex, and allows anti-fluorescein to quench the fluorescence by binding to the fluorescer. This phenomenon is the basis of a new homogeneous assay technique that requires no separation step. The steric exclusion of anti-fluorescein from fluorescein-labeled human IgG immune complexes was altered by changing the molecular dimensions ob both antifluorescein and the immune complex. The assay did not require highly purified fluorescein-labeled human IgG. An assay is demonstrated in which was used a fluorescein-labeled human IgG conjugate containing IgG that was only 10% pure. Measurement of IgG in human serum samples correlated well with results by radial immunodiffusion. The method is applicable to the assay of both proteins and analytes of low molecular mass.
D000936:Antigen-Antibody Complex; D005452:Fluoresceins; D005455:Fluorescent Antibody Technique; D006801:Humans; D007074:Immunoglobulin G / Q000032:analysis*; D007700:Kinetics
D000936:Antigen-Antibody Complex; D005452:Fluoresceins; D007074:Immunoglobulin G