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99) The fluorescent polarization immunoassay (FPIA) is a homogeneous assay for measuring low molecular weight molecules, such as drugs. In the quantification of a drug level by FPIA, which one of the following best explains how the non–antibody-bound fluorescent-labeled drug is differentiated from the antibody-bound fluorescent-labeled drug?

No fluorescence is observed when antibody binds to the fluorescent-labeled drug. Rotation of the non–antibody-bound fluorescent-labeled drug is slower than the antibody-bound fluorescent-labeled drug and depolarizes the emitted light. The antibody-bound fluorescent-labeled drug emits less polarized light than the non–antibody-bound fluorescent-labeled drug. The antibody-bound fluorescent-labeled drug emits polarized light. The non–antibody-bound fluorescent labeled drug depolarizes the emitted light. Rotation of the non–antibody-bound fluorescent-labeled drug is faster than the antibody-bound fluorescent-labeled drug and emits more polarized light.

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• FPIA is a homogeneous automated assay based on the amount of polarized fluorescent light detected when the fluorescent label is excited with polarized light.

• If a fluorescent molecule absorbs polarized light to produce the excited state, the emitted light will also be polarized if the fluorophore does not rotate during its time in the excited state. If the fluorophore rotates in the excited state before fluorescence emission takes place, the emitted light will be depolarized.

• The principle of FPIA involves competition between a fluorescent-labeled antigen (i.e., the drug; fluorescein is the most common fluorophore) and the antigen (i.e., free drug) in the sample for binding sites on an antigen-specific (i.e., drug-specific) antibody.

• The antibody-bound fluorescent-labeled antigen is a large molecule and rotates slowly in the excited state. Therefore, the polarization of the emitted light is not significantly changed from the polarized light that was used to excite the molecule.

• The non–antibody-bound fluorescent-labeled antigen is a small molecule and rotates rapidly in the excited state before fluorescence light is emitted. In this case, the emitted light is depolarized.

• In an FPIA assay, the amount of polarized light emitted is inversely related to the sample concentration of the analyte to be measured. At low analyte concentrations, more of the fluorescent-labeled antigen will bind to the antibody, resulting in a large amount of emitted polarized light.

Smith DS, Eremin SA: Fluorescence polarization immunoassays and related methods for simple, high-throughput screening of small molecules. Anal Bioanal Chem 2008;5:1499-1507.

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Clinical Pathology: General Principles, Lab Management, Clinical Chemistry

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