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.
Terpetschnig
E
E
Department of Biological Chemistry, University of Maryland at Baltimore, School of Medicine 21201, USA.
Szmacinski
H
H
Lakowicz
J R
JR
D015082:2,2'-Dipyridyl / Q000031:analogs & derivatives* / Q000737:chemistry; D000818:Animals; D001667:Binding, Competitive; D016231:Fluorescence Polarization Immunoassay / Q000379:methods*; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology; D007700:Kinetics; D008024:Ligands; D051379:Mice; D008956:Models, Chemical; D009942:Organometallic Compounds / Q000737:chemistry*; D012428:Ruthenium / Q000737:chemistry*; D012709:Serum Albumin / Q000032:analysis* / Q000276:immunology; D013997:Time Factors
D007074:Immunoglobulin G; D008024:Ligands; D009942:Organometallic Compounds; D012709:Serum Albumin; D015082:2,2'-Dipyridyl; C058014:ruthenium bis(bipyridine)dicarboxybipyridine; D012428:Ruthenium