Fluorescence polarization immunoassay polarized emission

The fluorescence polarization immunoassay is used for routine, automated immunoassay of small molecules, such as drugs. It depends on the principle that a fluorophore attached to a macromolecule such as an antibody is not free to rotate in solution. If polarized light is used to stimulate the fluorophore to fluoresce, emission from the bound fluorophore (attached to the antibody, which is bound to a surface) will continue to be polarized, but polarization will be lost from free fluorophore (111). 

The use of fluorescent labels has been very successful for inorganic complexes such as the europium chelates, which are used today in the Delfia commercial system. In contrast, fluorescence has rarely been employed in the area of organometallic tracers, and only the immunoassay developed by Lakowicz uses such a tracer [88]. This is a homogeneous competitive immunoassay, the tracer being the complex (Re-L) -HSA, 48 obtained as shown in Scheme 8.20 and the detection method fluorescence polarization (FP). This compound 48 displays highly polarized emission (with a maximum polarization near 0.4 and maximum anisotropy near 0.3) in the absence of rotational diffusion and a long average lifetime (2.7 ps) when bound to proteins in air-equilibrated aqueous solution. 

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