The use of disulfonatonaphthalimide fluorescent dyes for the fluorescence polarization immunoassay of steroids

A series of fluorescent disulfononaphthalimide (lucifer yellow) derivatives of testosterone and estriol have been synthesized: testosterone-3- and estriol-6-ureidooxime-lucifer yellow, testosterone-3-and estriol-6-(amidoureido)methoxyoxime lucifer yellow and testosterone-3-amidoheptylaminoethylsulfonylphenyl methoxyoxime lucifer yellow. The fluorescence lifetimes of these derivatives were higher than those of the free dye while the quantum yields were of the same order. The compounds were therefore compared in terms of their utility for steroid fluorescence polarization immunoassay. Because of the high Stokes’ shift of these derivatives the blanks due to diffusion polarization were considerably reduced relative to those usually obtained with fluorescein derivatives. The sensitivity of the immunoassays were therefore improved and, with testosterone-3-(amidoureido)methoxyoxime lucifer yellow, the optimum of the testosterone derivatives, it was possible to assay 0.3 to 25 ng/ml testosterone. The use of monoclonal antibodies and simplification of the instrumentation should permit the development of clinically useful assays.