Immunoassay fluorescence excitation transfer

E. F. Ullman, M. Schwarzberg, and K. E. Rubenstein, Fluorescent excitation transfer immunoassay. A general method for determination of antigens, J. Biol. Chem. 251, 4172 4178 (1976). 

E. F. Ullman and P. L. Khanna, Fluorescence excitation transfer immunoassay (FETI), Methods in Enzymology 74, 28-60 (1981). 

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. 

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. 

The original methods of fluorescence exciting transfer immunoassay (Ul) and enzyme channeling immunoassay (L9) are similar to the proximal linkage immunoassay. In the former, two reactants are coupled to solid antigen and antibody and, in the latter, they are coupled to two kinds of monoclonal antibodies. The enzyme enhancement immunoassay (G4) also falls in this category. 

In a heterogeneous sandwich immunoassay employing fluorescence excitation transfer (Eq. 6.7 and Fig. 6.6), draw the plots of emission intensity versus log[Ag] that would be expected if emission from (a) FI and (b) F2 were measured. 

An alternative homogenous fluorescence-based detection system is fluorescence resonance energy transfer (FRET). This phenomenon occurs when two fluorophores are in close proximity, and the donor fluorophore has an emission spectrum that overlaps the excitation spectrum of the acceptor fluorophore. When the donor fluorophore is excited, energy is transferred from donor to acceptor with the result that the intensity of emission from the donor is reduced (quenched). If both the analyte and antibody are coupled to fluorophores with overlapping spectra, then FRET will occur only when the complex forms. Thus, FRET has not been applied widely in conventional immunoassays. 

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