Mechanisms of fluorescence quenching in donor-acceptor labeled antibody-antigen conjugates

The cyanine dyes Cy5 and Cy5.5 are presented as a new long wavelength-excit able donor-acceptor dye pair for homogeneous fluoroimmunoassays. The deacti vation pathways responsible for the quenching of the fluorescence of the an tibody-bound donor are elucidated. Upon binding of the donor dye to the ant ibodies at low dye/protein ratios, its fluorescence quantum yield rises to unity. Higher dye/protein ratios lead to progressive aggregation of the dye s, which results in quenching of monomer fluorescence due to resonance ener gy transfer (RET) from the monomers to the nonfluorescent dimers. The depen dence of the quenching efficiency on the labeling ratio is described quanti tatively by assuming a Poisson distribution of the dyes over the antibodies . The maximum fluorescence intensity per antibody is obtained at a labeling ratio of 4. Upon formation of the antibody-antigen complex, electron trans fer and RET to the antigen-bound acceptor dye occur. Steady-state and time- resolved fluorescence measurements reveal that approximately 50% of the don or quenching is due to RET, while the residual quenching effect is caused b y the static quenching process.