Fluorescence polarization immunoassay (FPIA) is a class of in vitro biochemical test used for rapid detection of antibody or antigen in sample. FPIA is a competitive homogenous assay, that consists of a simple prepare and read method, without the requirement of separation or washing steps.
The basis of the assay is fluorescence anisotropy, also known as fluorescence polarization. If a fluorescent molecule is stationary and exposed to plane-polarized light, it will become excited and consequently emit radiation back to the polarized-plane. However, if the excited fluorescent molecule is in motion (rotational or translational) during the fluorescent lifetime, it will emit light in a different direction than the excitation plane. The fluorescent lifetime is the amount of time between the absorption moment and the fluorescent emission moment.
Typically, the rate at which a molecule rotates is indicative of its size. When a fluorescent-labelled molecule (tracer) binds to another molecule the rotational motion will change, resulting in an altered intensity of plane-polarized light, which results in altered fluorescence polarization. Fluorescence polarization immunoassays employ a fluorophore bound antigen that when bound to the antibody of interest, will increase fluorescence polarization. The change in polarization is proportional to the amount of antigen in sample, and is measured by a fluorescence polarization analyzer.