Detecting antigens by fluorescence immunoassay, useful e.g. for diagnosis, by performing both competitive and sandwich assays to cover a broad concentration range

摘要

Method for detecting an antigen (Ag) by fluorescence immunoassay using two Ag-specific antibodies (Ab), one (Ab1) being labeled with a fluorophore (I) and the other (Ab2) immobilized. The amount of bound Ag is determined by evanescent wave excitation and measurement of fluorescence. A known amount of Ab1 is mixed with sample containing Ag (which has more than one binding site), so that it is present in excess over Ag, and the reaction mixture incubated with support-bound Ab2, to capture the Ab1-Ag complex formed. The amount of Ag bound is determined (sandwich assay). Residual free Ab1 is then incubated with a solid phase that carries Ag, so that it becomes bound, and measurement of bound Ag repeated for this support (competitive assay). The Ag concentration is then determined from the results of both sandwich and competitive assays.