Fluorescence polarization: an analytical tool for immunoassay and drug discovery

Fluorescence polarization (FP) is an intrinsically powerful technique for the rapid and homogeneous analysis of molecular interactions in biological/chemical systems. The technique has been successfully used to diagnose various viral and infectious diseases in humans and animals, to monitor therapeutic drug levels and substances of abuse in body fluids and to determine food born pathogens, grain mycotoxins and pesticides. It has also been used in monitoring enzyme catalyzed hydrolysis, protein-protein interactions, DNA diagnostics and high throughput screening during the course of drug discovery. Work by various groups, including our own, have demonstrated that the technique can replace a substantial number of solid phase assays. FP, defined by the equation P = [IV - IH] / [IV + IH] (where V and H are the vertical and horizontal components of the intensity I of emitted light respectively when exited by vertically plane polarized light), is independent of the intensity of the light and the concentration of the fluorophore. Hence it is functional in colored and cloudy solutions. The FP of a fluorophore is proportional to its rotational relaxation time, which in turn depends upon its molecular volume (or molecular weight) at constant temperature and solution viscosity. When a fluorophore-labeled ligand binds to a larger molecule, equilibrium is established rapidly and the FP increases. This property has been successfully exploited in many fields as described in this review.