Homogeneous model immunoassay of thyroxine by phase-modulation fluorescence spectroscopy

We describe a homogeneous competitive model immunoassay for determination of thyroxine by multifrequency phase-modulation fluorescence. Using a nonradiative energy transfer transduction mechanism, B-phycoerythrin conjugated to thyroxine is the energy donor and a carboxymethylindocyanine dye conjugated to anti-thyroxine antibody is the energy acceptor. Energy transfer from B-phycoerythrin to the acceptor results in a decreased lifetime and/or phase angle. The fluorescence lifetime change reflects the extent of energy transfer. In the competitive immunoassay format, the donor-thyroxine conjugate and an analytical sample of thyroxine compete for acceptor-antibody binding sites, resulting in a phase angle change which is dependent on the amount of thyroxine in the sample. Dose-response curves of phase angle versus thyroxine concentration are comparable to steady-state intensity curves. Since phase-modulation lifetime measurements are largely independent of total signal intensity, sources of optical interference are minimized. The potential for whole blood measurements exists since the energy transfer lifetime method can be extended to longer wavelengths.