Introduction to FPIA

April 2003; updated August 2004

Fluorescence Polarization (FP) assays are homogeneous, single-step assays ideally suited for high-throughput screening (HTS) of large numbers of samples. All FP assays employ a large molecular species, or binding partner (BP), in conjunction with a small, low molecular weight fluorophore-labeled analyte (FA). When the large BP molecule is an antibody, the assay is referred to as a fluorescence polarization immunoassay (FPIA).

Fluorescence is by definition the ability of a molecule to absorb the energy of an incoming (excitation) photon and then re-emit most of this energy as a new, slightly less energetic (emission) photon.

A small fluorescent molecule will rotate appreciably during the very small interval of time between absorption of a photon and emission of the fluorescence photon.

If the excitation light is polarized, this rotation will result in complete randomization of the plane of the emitted light. Thus, small fluorescent molecules depolarize an excitation pulse of polarized light (well #1).

Large fluorescent molecules (MW > 100,000) do not rotate appreciably in the same small interval of time. They will therefore emit light that retains some of the polarization of the polarized excitation light. This polarization is quantified as milli-polarization units, or mP. A fluorescence polarization reader is required to make this measurement.

When a small fluorescent molecule becomes tightly bound to a large one, as in the binding of FA to an antibody, the rotational speed of the small molecule is abruptly reduced to that of the entire complex as a whole (well #2). Therefore, FA bound to its antibody represents a large fluorescent molecule, which exhibits a high degree of fluorescence polarization (FP).

FPIAs are based on the competition of FA with free (i.e. unlabeled) analyte in the samples or standards for the high affinity binding site an antibody. A microplate well filled with the FA:antibody complex will give a high FP reading. Addition of a increasing amounts of unlabeled analyte will result in a competition between the unlabeled FA for the antibody.

As the competition happens, some of the FA will be released from the antibody, and will resume its intrinsic, rapid rate of rotation. This will cause a detectable loss of FP in the well (well #4).

The addition of large amount of analyte will result in a much larger reduction in the mP of the well (well #5). Plotting mP versus analyte concentration allows the construction of a standard curve with a broad dynamic range. This is similar to, but not strictly analagous to, the sigmoidal dose-response curve in a traditional solid phase EIA.