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....

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Bibliographic Details
Published in:Analytical biochemistry 1995-05, Vol.227 (1), p.140-147
Main Authors: Terpetschnig, E., Szmacinski, H., Lakowicz, J.R.
Format: Article
Language:English
Subjects:
2,2'-Dipyridyl - analogs & derivatives
2,2'-Dipyridyl - chemistry
Animals
Binding, Competitive
Fluorescence Polarization Immunoassay - methods
Humans
Immunoglobulin G - immunology
Kinetics
Ligands
Mice
Models, Chemical
Organometallic Compounds - chemistry
Ruthenium - chemistry
Serum Albumin - analysis
Serum Albumin - immunology
Time Factors
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Summary: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.
ISSN:0003-2697
1096-0309
DOI:10.1006/abio.1995.1263