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.