Anisotropy fluorescence polarization immunoassay

The final type of immunoassay is the fluorescence polarization immunoassay (FPI). Assays of this type are based on anisotropy measurementsof labeled antigens. The use of the term polarization instead otsoisotropy is historical, and now entrenched In the liftrature. The anisotropy of a mixture (r) is determined by the anisotropies of the... 

Fluorescence polarization immunoassays (FPIs) are widely used to measure the amounts of drugs (D) and small molecides in chnical samples (Chapter 19). FPIs are based on the changes in polarizadon (or anisotropy) which occur when a labeled < g analog (F-D) binds to an antibody specific filT that drag O igure 20.20). The anisotropy of the lafaded drag can be estiinated fiom the Perrin equation. 

Figure 35 (a) Principle of a fluorescence polarization immunoassay and (b) the Perrin equation defining the expected anisotropy (r),... 

The versatility of luminescence goes beyond intensity-, wavelength- and kinetic-based measurements. Fluorescence polarization (or anisotropy) is an additional parameter still largely unexplored for optical sensing yet widely used in Biochemistry to study the interaction of proteins, the microfluidity of cell membranes and in fluorescence immunoassays. Although only a few optosensors based on luminescence polarization measurements can be found in the literature, elegant devices have recently been reported to measure chemical parameters such as pFI or O2 even with the bare eye41. 

Anotiier ratiometric mediod is based on die measurement of fluorescence polarization or anisotropy. In this case the analyte causes a change in the polarization of the label. Anisotropy measurements are frequently used in competitive immunoassays, in which the actual analyte displaces labeled analyte that is bound to specific antibody. This results in a decrease in the anisotropy. Anisotropy values are calculated using the ratio of polarized intensity measurements. The use of an intensity ratio makes the anisotropy measurements independent of fluorophm c(mi-centration as long as the measuremeDts are not distorted by autofluoresccnce or pow signal-to-noise ratio. Polarization immunoassays are discussed in Section 19.9X). 

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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