FPIA quantifies the change in fluorescence polarization of reaction mixtures of fluorescent-labelled tracer, sample antigen, and defined antibody. Operating under fixed temperature and viscosity allows for the fluorescence polarization to be directly proportional to the size of the fluorophore. Free tracer in solution has a lower fluorescence polarization than antibody-bound tracer with slower Brownian motion. The tracer and the specific antigen will compete to bind to the antibody and if the antigen is low in concentration, more tracer will be bound to the antibody resulting in a higher fluorescence polarization and vice versa.
A conventional FPIA follows the procedure below:
# A specific quantity of sample is added to reaction buffer.
# The solution is allowed to equilibrate at room temperature for approximately two minutes.
# The solution is evaluated in a fluorescence polarization analyzer to gather a baseline measurement.
# A specific quantity of antigen conjugated with fluorophore is added to the solution.
# The solution again equilibrates for approximately two minutes.
# The solution is evaluated again by the fluorescence polarization analyzer.
# The fluorescence polarization value for the tracer containing solution is compared to the baseline and magnitude of difference is proportional to quantity of target analyte in sample.