Fluorescence Energy Transfer Immunoassays

An immunosensor based on a competitive fluorescence energy-transfer immunoassay was reported by Anderson 105) for the measurement of phenytoin. Texas red-labeled antibody was incubated with a phenytoin derivative. On displacement of the derivative by the antigen, the change in the fluorescence signal was recorded. Detection limits approached 5 /iM with response times ranging from 5 to 30 min. 

M. Plebani and A. Burlina, Fluorescence energy transfer immunoassay of digoxin in serum, Clin. Chem. 31, 1879-1881 (1985). 

Fiber-optic sensors based on controlled-release polymers provide sustained release of indicating reagents over long periods. This technique allows irreversible chemistries to be used in the design of sensors for continuous measurements. The sensor reported in this paper is based on a fluorescence energy transfer immunoassay. The sensor was cycled through different concentrations of antigen continuously for 30 hours. 

Figure 10. Proposed Adaptation of a Fluorescence Energy Transfer Immunoassay to the Microparticle Sensor Design. A mixture of two different microparticles, each containing different reagents, are entrapped physically in the polyacrylamide layer. The reagents released from the microparticles set up a competition reaction between the free and labeled antigens for the available binding sites of labeled-antibody. The immunocomplexes formed have different emission spectra, allowing quantitation of free antigen concentration.

Sq635-b and Sq660 were also utilized as donor-acceptor pairs in combination with an HSA/anti-HSA system, in a fluorescence energy transfer (FRET)-based immunoassay [95, 96]. 

Oswald B, Gruber M, Bohmer M, Lehmann F, Probst M, Wolfbeis OS (2001) Novel diode laser-compatible fluorophores and their application to single molecule detection, protein labeling and fluorescence resonance energy transfer immunoassay. Photochem Photobiol 74 237-245... 

Khanna, P.L., and Ullman, E.F. (1980) 4, 5 -dimethoxy-6-carboxyfluorescein A novel dipole-dipole coupled fluorescence energy transfer acceptor useful for fluorescence immunoassays. Anal. Biochem. 108,156. 

J. R. Lakowicz and B. P. Maliwal, Fluorescence lifetime energy transfer immunoassay quantified by phase-modulation fluorometry, Sensors andActuators B 12, 65-70 (1993). 

The immunoassay selected is based on a competitive fluorescence energy transfer mechanism. This type of assay is general and can be adapted to any antibody and antigen system. The competing reactions are given in equation (1) and (2) ... 

The first obvious use of FRET is in sandwich immunoassay. A first report along this line was presented by Ullman et al. in 1976 [168]. Wei et al. investigated 10 combinatorial pairs of conventional fluorescent dyes and assessed the optimal conditions for energy transfer [169]. Oswald et al. described a fluorescence resonance energy transfer immunoassay for human serun albumin (HSA) based on the indo-lium dye Sq635 (Fig. 14.10) and the benz[e]indolium dye Sq660 [83]. A detection limit of 10 M of HSA was reported. 

Schobel, U., Egelhaaf, H. J., Brecht, A., Oelkrug, D. and Gauglitz, G. (1999). New donor-acceptor pair for fluorescent immunoassays by energy transfer. Bioconjug. Chem. 15, 1107-14. 

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