Homogeneous fluorescence polarization immunoassay

Like in RILAs, an advantage of fluorescence detection is the possibility of developing homogeneous FILAs using direct or indirect (competitive or displacement) approaches. The fluorescence polarization immunoassay (PFIA) and their homolog fluorescence polarization immuno-like assay (PFILA) are two of the most widely used procedures in homogeneous fluoroassays. Both are based on the principle that fluorescence polarization gives a direct measure of the bound/free ratio of the labeled analyte (tracer) without the need for their separation [23, 28]. 

Fluoroimmunoassay These assays rely on the use of fluorescence-emitting labels. Typically, antibodies labelled with afluorophore are used, for example, fluorescein isothiocyanate. After washing, measured fluorescence relates to the amount of antigen. Another useful variant is called the homogeneous fluorescent polarization immunoassay that is commonly used to measure dmgs and analytes. 

Nonisotopic methods have also been described. For example, a homogeneous (nonseparation) fluorescence polarization immunoassay for DHEA-S that uses a rabbit polyclonal antibody and a DHEA-fluorescein tracer is available. The measured polarization is inversely related to DHEA-S concentration. This fully automated system has a dynamic range of 1 to lOOOjJ-g/dL (0,03 to 27 Limoi/L), and interassay coefficients of variation are less than 10% over a broad concentration interval (25 to lOOOpg/dL 0.7 to 27pmol/L). Assay time is about 15 minutes for a single sample and 30 minutes for 20 samples. 

These elegant techniques do not require separation of the bound and unbound fractions prior to measurement of response. Such assays offer advantages by simplifying and eliminating a step, but are more prone to matrix effects. An example of a homogeneous assay is fluorescence polarization immunoassay (FPIA, Figure 4.28). ... 

Figure 4.28 Fluorescent polarization immunoassay (FPIA), a homogeneous assay.The -F indicates a fluorescing iabel. 

Fluorescent polarization immunoassay A homogenous immunoassay in which the label is a fluo-rophore. 

Other successful homogeneous assays have been developed such as the fluorescence polarization technique made available by Abbott Laboratories of North Chicago, USA. This competitive immunoassay technique relies upon the principle... 

Methods for the Detection of Antigens/Antibodies Equilibrium and kinetic inhibition assays based upon fluorescence polarization, 70, 3 fluorescence excitation transfer immunoassay (FETI), 70, 28 indirect quenching fluoroimmunoassay, 70, 60 the homogeneous substrate-labeled fluorescent immunoassay, 70, 79 fluorescence immunoassays using plane surface solid phases (FIAPS), 70, 87. 

Tachi T, Kaji N, Tokeshi M, Baba Y (2009) Microchip-based homogeneous immunoassay using fluorescence polarization spectroscopy. Lab Chip 9 966-971... 

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. 

Heterogenous fluorescence immunoassays can be carried out with the aid of the same separation procedures used in radioimmunoassay. The more expedient homogenous fluorescence immunoassays require quenching, enhancement, polarization, or shifting of the fluorescence of the label upon binding of the labeled analyte to its antibody. Occasionally, a second antibody, directed at the anti analyte antibody, will be used in a double-antibody method to precipitate the bound labeled and unlabeled analyte or to alter the optical properties of the label in such a way as to make the analysis more sensitive. 

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