Homogeneous immunoassay fluorescent labels

In the past ten years, numerous applications of fluorescence methods for monitoring homogeneous and heterogeneous immunoassays have been reported. Advances in the design of fluorescent labels have prompted the development of various fluorescent immunoassay schemes such as the substrate-labeled fluorescent immunoassay and the fluorescence excitation transfer immunoassay. As sophisticated fluorescence instrumentation for lifetime measurement became available, the phase-resolved and time-resolved fluorescent immunoassays have also developed. With the current emphasis on satellite and physician s office testing, future innovations in fluorescence immunoassay development will be expected to center on the simplification of assay protocol and the development of solid-state miniaturized fluorescence readers for on-site testing. 

Ericsson, 1990), the synthesis of fluorescently labeled DNA probes (L. M. Smith et al., 19 8 5), as a label in homogeneous immunoassay systems (Nithipatikom and McGown, 1987), to investigate specific interactions of proteins with cells surfaces (Hochman et al., 1988), and as an important fluorescent tag of antibodies in immunohistochemical staining techniques (Davidson and Hilchenbach, 1990). 

The term homogeneous immunoassay may be defined as an immunoassay in which the extent of the antigen-antibody reaction can be determined without physical separation of the free and bound forms. This term is usually used for immunoassays such as enzyme and fluorescence immunoassays in which labeled substances (markers) are used. It does not include immunoassay systems such as nephelometry in which no labeled substances are used. Homogeneous immunoassays are widely used as routine tests because the procedures involved are simple. The principle of homogeneous immunoassay is based on changes in signals of the indicators by the antigen-antibody reaction (N5, U2). 

In this assay, marker-labeled antigen itself acts as substrate. Kohen et al. (K9) developed a homogeneous immunoassay for steroid using a steroid-fluorescent dye conjugate that yields fluorescent products upon hydrolysis with enzyme. The steroid-fluorescent dye conjugate is inactive as a substrate when bound to the antibody to steroid [F-Ag Ab]. But when unlabeled steroid [Ag] is added, it binds competitively to the antibody [Ab Ag],. and the free form of steroid-fluorescent dye conjugate [F-Ag], which is... 

Among homogeneous fluoroimmunoassays based on conventional fluorimetry, substrate-labeled fluoro-immunoassay is another alternative for both hapten and protein determination using a fluorescent label. As in other homogeneous competitive immunoassays, the sensitivity is limited by the serum background fluorescence and the limited amount of tracer that can be used. 

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. 

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

Figure 6.2. Chemical structure of 8-[3-(7-/)-galactosylcoumarin-3-carboxyamido )propyl ] theophylline (B) used in the homogeneous substrate-labeled fluorescent immunoassay for theophylline (A). (Reprinted from Ref. 3, with permission from the American Association for Clinical Chemistry.)...

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. 

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]. 

B6. Burd, J. F., Wong, R. C., Feeney, J. E., Carrico, R. J., and Boguslaski, R., Homogeneous reactant-labeled fluorescent immunoassay for therapeutic drugs exemplified by gentamicin determination in human serum. Clin. Chem. 23, 1402-1408 (1977). 

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. 

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