Polarization immunoassays using reactive ruthenium metal-ligand complexes as luminescent labels

The first competitive fluorescence polarization immunoassay using ruthenium metal-ligand complexes (5-MC and 55-DC) as labels is described. These were newly synthesized and characterized in terms of their spectra, their covalent linkage to proteins, and their use in both homogeneous and competitive immunoassays. Linkage to proteins was achieved by the N-hydroxysuccinimide ester method, which was demonstrated for the systems HSA-anti-HSA and myoglobin-anti-myoglobin. The values of the fundamental polarization are 0.18 for 5-MC and 0.33 for 55-DC. Polarization immunoassays with labeled HSA and myoglobin were performed in the homogeneous format and resulted in an increase of the fluorescence polarization of up to 100%. In the competitive assay, a decrease in polarization of >90% was detectable. When the competitive HSA immunoassay was validated against an independent test both methods gave almost the same results.