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-excitable donor-acceptor dye pair for homogeneous fluoroimmunoassays. The deactivation pathways responsible for the quenching of the fluorescence of the antibody-bound donor are elucidated. Upon binding of the donor dye to the antibodies at low dye/protein ratios, its fluorescence quantum yield rises to unity. Higher dye/protein ratios lead to progressive aggregation of the dyes, which results in quenching of monomer fluorescence due to resonance energy transfer (RET) from the monomers to the nonfluorescent dimers. The dependence of the quenching efficiency on the labeling ratio is described quantitatively 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 transfer and RET to the antigen-bound acceptor dye occur. Steady-state and time-resolved fluorescence measurements reveal that approximately 50% of the donor quenching is due to RET, while the residual quenching effect is caused by the static quenching process.