Fluorescence Polarization Immunoassays

Fluorescence polarization immunoassays (FPIA) are quick and efficient homogeneous mix-and-read assays. The principle of the FPIA is similar to that of the competitive ELISA. Both assays work on the principle of small molecule competitive binding where quantification is based on antibody equilibrium binding to free antigen and conjugate. As the amount of antigen increases, the amount of conjugate bound to antibody decreases leading to a detection response that is inversely proportional to antigen concentration.

In Enzo Life Sciences’ FPIA kits, the conjugate is covalently attached to a fluorescence tag, the aromatic molecule fluorescein. All binding events in an FPIA take place in free motion whereas the ELISA utilizes solid phase binding. The units used to quantify FPIA are the milli-polarization unit (mP). When excited by polarized light, the conjugate molecule will emit light of a lower energy state. Molecules are in motion, which results in the non-specific directional emission of light. The faster the molecule tumbles, the more depolarized the light. Therefore, conjugate bound to antibody will tumble more slowly due to its size, which will yield a greater amount of polarized light and a larger mP. This is how bound conjugate molecules are differentiated from unbound conjugate which has been displaced by analyte in the standard or sample.