What occurs in a Fluorescence Polarization Immunoassay (FPIA) with increased drug sample levels?

In a Fluorescence Polarization Immunoassay (FPIA), the principle behind the assay is based on the measurement of the rotation of fluorescently labeled molecules in solution. When a sample containing a drug is introduced into the system, the drug can compete with a fluorescently labeled tracer for binding sites on an antibody.

As the concentration of the drug in the sample increases, a greater number of drug molecules will bind to the antibody, effectively displacing the fluorescent tracer. This displacement leads to a decrease in the overall polarization of the fluorescent signal. The higher presence of free drug molecules results in more rapid rotation of the fluorescent label because the bound fraction (the tracer attached to the antibody) is reduced. Consequently, the resulting decrease in polarization reflects this high drug concentration.

Thus, in FPIA, increased drug sample levels correspond to a decrease in polarization. This relationship allows for quantifying the concentration of the drug based on the degree of fluorescence polarization, forming the basis for the assay's diagnostic capabilities.