Time-resolved detection of energy transfer: theory and application to immunoassays.

Energy-transfer measurements based upon acceptor fluorophore emission are plagued with background fluorescence resulting from absorption of the excitation light by the acceptor fluorophore. The present work examines the use of a long-lifetime donor fluorophore and a short-lifetime acceptor fluorophore, combined with pulsed-laser excitation and electronic gating of detector signals, to separate the component of acceptor emission due to energy transfer from the component due to absorption of the excitation light. Theoretical equations describing the acceptor fluorescence and integrated acceptor fluorescence show that increasing the integration delay relative to the excitation pulse should greatly enhance detection of the energy-transfer component. The time-resolved detection of energy transfer was tested in a competitive immunoassay format in which antibodies to human immunoglobulin G (IgG) F(ab')2 fragments were covalently labeled with pyrenebutyrate (tau = 100 ns) and IgG Fab' fragments were covalently labeled with B-phycoerythrin (tau = 2.5 ns). Solutions containing these two conjugates exhibited energy transfer from the pyrenebutyrate to the B-phycoerythrin upon excitation with a nitrogen laser. Acceptor emission was measured with 0- and 20-ns integration delays and the ratios of the energy-transfer component to the laser-excited component were found to increase by 9- to 15-fold when the 20-ns delay was used in three series of immunoassays. Good agreement between the experimental data and theory was obtained following convolution of the theoretical fluorescence responses with the instrumental response of the fluorometer.