In fluorescence polarization immunoassay, which statement is true?

• A. Both antigen and antibody are labeled.
• B. Large molecules polarize more light than smaller molecules.
• C. When binding occurs, there is quenching of the fluorescent tag.
• D. The amount of fluorescence is directly proportional to concentration of the analyte.

Answer: (B)

Molecular rotation during the fluorescence lifetime determines how polarized the emitted light remains. Larger molecules rotate more slowly, so the emitted light stays more polarized; small molecules rotate quickly and depolarize the emission. In a fluorescence polarization immunoassay, a fluorescent tracer (the antigen) binds to a larger antibody. Free tracer is small and tumbles rapidly, giving low polarization, while when bound to the antibody the complex is bigger and rotates more slowly, producing higher polarization. Therefore, large molecules polarize more light than smaller ones. The other statements don’t reflect this key mechanism: usually only the tracer is labeled, quenching isn’t the primary effect here, and the readout is polarization rather than fluorescence intensity proportional to analyte concentration.