The tag should have a strong absorption at the excitation wavelength of the experiment.
The quantum yield of the labeled species should be large.
The absorption and emission bands should be in the visible range to avoid interference from native proteins.
The separation between excitation and emission peaks should be large.
A further requirement of any immunoassay method is the possibility of distinguishing between free and bound labeled ligand, or the two species must be separated somehow. Here, FIA has a distinct advantage over RIA: by monitoring fluorescence polarization, the separation step is circumvented.
In fluorescence polarization, the excitation light is passed through a polarizing prism, and the emitted light is analyzed with another polarizer that is alternately oriented parallel and perpendicular to the excitation polarizer. The amount of polarization retained by the molecule depends upon its Brownian rotation between absorption and emission. A bound ligand will retain much of its polarization because the large molecular volume of the antibody gives the complex a long rotational relaxation time. The free ligand, on the other hand, has a much smaller volume, so the rotational relaxation time is shorter than its fluorescence lifetime. The emitted radiation, therefore, has a polari