Fluorescence quenching with immunological pairs in immunoassays

Abstract

Immunoassays are provided employing antibodies and a fluorescer-quencher (F-Q) chromophoric pair, wherein one or both of the chromophoric pair are bonded to antibodies. Depending on the particular ligand of interest, or whether antibodies are to be assayed, various reagent combinations can be employed, where the amount of quenching is directly related to the amount of ligand or antibody present in the assay medium. In carrying out the assay for ligands, the unknown and antiody specific for the ligand of interest to which is bound one of the F-Q pair, are combined in an aqueous buffered medium. Depending on the protocol, different assay reagents are employed in the aqueous buffered medium: (1) ligand analog bonded to the other of the F-Q pair; (2) antiobodies specific for the ligand to which is bound the other of the F-Q pair or; finally, (3) a combination of a plurality of ligands bonded together through linking groups to a hub molecule, usually a polymer, in combination with antibody bound to the other of the F-Q pair. The fluorescar is electronically excited, for example by irradiation with light at a wavelength, absorbed by the fluorescing molecule and the amount of emitted light determined. By employing appropriate standards, the presence and amount of the ligand can be determined. The technique for the determination of antibodies in a competitive mode is substantially the same except that ligand is added as a reagent.