Fluorescence excitation transfer

Fluorescence Excitation Transfer. This modified-sandwich method (shown in Eq. 6.7) has both antibodies labeled, but with different fluorophores (FI and F2) that are chosen so that the emission spectrum of one overlaps with the excitation spectrum of the other. Internal quenching thus occurs when FI and F2 are held in close proximity. If FI and F2 are close, the emission of FI is quenched 

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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. 

Figure 6.6. Fluorescence excitation transfer in a two-site sandwich assay.

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. 

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