FLUORESCENCE POLARIZATION IMMUNOASSAY OF A HIGH-MOLECULAR-WEIGHT ANTIGEN BASED ON A LONG-LIFETIME RU-LIGAND COMPLEX

We describe a new class of fluorescence polarization immunoassays based on the luminescence from an asymmetrical Ru-ligand complex. We found that such a complex displays larger polarization values than those of comparable symmetrical complexes and appear to be highly photostable in aqueous solution, We synthesized a conjugatable Ru-ligand complex, which was used to label human serum albumin (HSA) as the antigen, The Ru-ligand complex displays a long decay time near 400 ns when covalently linked to proteins. We found that the steady-state polarization of labeled HSA was sensitive to binding of anti-HSA, resulting in a 200% increase in polarization, The labeled HSA was also used in a competitive format using unlabeled HSA as the antigen, The time-resolved anisotropy decays demonstrate increased correlation times for labeled HSA in the presence of anti-HSA, an effect which was partially reversed in the presence of unlabeled HSA, These results demonstrate the potential of the metal-ligand complexes to be used in the fluorescence polarization immunoassay of high-molecular-weight analytes, The use of such metal-ligand complexes enable fluorescence polarization immunoassays which bypass the usual limitation to low-molecular-weight antigens, which is a consequence of the 2-5 ns decay time of the previously used fluorophores. (C) 1995 Academic Press,Inc.