Fluorescence polarization immunoassay of a high-molecular-weight antigen using a long wavelength-absorbing and laser diode-excitable metal-ligand complex.

We describe a fluorescence polarization immunoassay (FPI) based on an osmium metal-ligand complex, Os(bpy)2(dcbpy). The Os complex was selected for its long wavelength absorption, which allows excitation with wavelengths up to 720 nm. At these wavelengths tissue absorbance and autofluorescence are minimal, and excitation can be accomplished with LEDs or laser diodes. The Os complex displays a lifetime of 19 ns and a high initial anisotropy at two excitation wavelengths, 505 and 690 nm. A reactive NHS-ester of Os- (bpy)2(dcbpy) was synthesized containing a NHS ester, and used to label the human serum albumin (HSA) or anti-HSA. The FPIs of HSA were performed with monoclonal and polyclonal antibodies using excitation at 505 or 685 nm. The results showed the potential of the Os-ligand complex to be used as a long excitation wavelength FPI probe for potential use in homogeneous immunoassays with simple excitation sources.