Fluorescence energy transfer immunoassay based on a long-lifetime luminescent metal-ligand complex

We describe an immunoassay based on fluorescence resonance energy transfer (FRET). The antigen was human serum albumin (HSA), which was labeled with a ruthenium-ligand complex, [Ru(bpy)2(phen-ITC)]2+. The antibody (IgG) to HSA was labeled with a nonfluorescent absorber, Reactive Blue 4. Association of the Ru-labeled HSA with the antibody was detected by three spectral parameters, a decreased quantum yield of Ru-HSA, a decrease in its fluorescence lifetime, and an increase in its fluorescence anisotropy. The steady-state anisotropy of Ru-HSA increased approximately eightfold upon binding to the antibody. These spectral effects were observed both in the direct association of the Ru-HSA with Reactive Blue 4-labeled antibody, and in a competitive assay format wherein unlabeled HSA competed with Ru-HSA for the binding sites on the antibody. Some nonspecific interactions of HSA may have occurred with Reactive Blue 4-labeled AHA, a difficulty which can be avoided with a different acceptor. The use of FRET provides a reliable means to alter the spectral properties upon antigen-antibody binding. The advantages of a ruthenium-ligand fluorophore include its long-wavelength absorption and emission, long fluorescence lifetime, and high photo-stability. Long wavelengths minimize problems of autofluorescence from biological samples, and long life-times allow off-gating of the prompt autofluorescence.