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Fluoresence polarization immunoassay (FPIA) is also a homogenous competitive immunoassay. In this system, fluorescein-labeled drug competes with unlabeled drug from the patient's serum sample for binding sites on an antibody reagent.

The patient's sample, presumably containing the therapeutic drug that is being monitored, and the fluorescein-labeled drug are added to a chamber containing antibody for that drug.

The labeled and unlabeled drug will compete for binding sites on the antibody. The greater the amount of drug in the sample, the fewer the number of binding sites that are available for the labeled analyte, leaving a greater number of small, free fluorescein-labeled molecules in the solution.

When the chamber is excited with plane polarized light, fluorescein will absorb the light and emit it at a higher wavelength as fluorescent light.

A small, free fluorescein-labeled drug rotates randomly and faster than it would if it were bound to antibody, interrupting the light and leading to less emission of light.

The larger antibody-drug-fluorescein complexes rotate slower and emit more light in the measured plane.

A lower level of drug in the patient's sample results in greater emission of polarized light because there are more antibody-drug-fluorescein complexes present to produce light in the measured plane. A higher level of drug in the patient's sample results in a lower emission of polarized light. This inverse relationship between the concentration of the drug and the polarization units (signal) is illustrated in the image below.
FPIA assays are rapidly being replaced by higher-throughput chemiluminescent assays. FPIA is now rarely used.