Fluorescein labeling of estrogen for application of fluorescence polarization binding assays for antibody and receptor

Abstract

The fluorescence polarization binding assay (FPBA) using fluorescein-labeled estrogen tracer is a homogeneous assay applicable to both estrogen antibody and estrogen receptor-binding assays. Two estrogen-ethylendiamine fluoresceinthiobamyl (E-EDF) tracers were synthesized; estrogen-6-EDF (E-6-F) derived from 6-ketoestradiol 6-( o-carboxymethyl) oxime and estrogen-17-EDF (E-17-F) was from 17β-estradiol 17-hemisuccinate. In both FPBAs using antibody and receptor, E-6-F tracer (Rf 365nm=0.58) showed a better binding response than E-17-F (Rf 365nm=0.70) indicating that the 17-position of estrogen seems to play an essential role as a binding site for antibody or receptor. In the optimized conditions of FPBA for E 2 using E-6-F tracer, antibody binding (K d=9.4×10 −9 M) is 50 times sensitive than receptor binding (K d=4.6×10 −8 M). Binding responses of estrogen and its related chemicals by FPBA indicate that antibody binding assay is able to screen the structural similarity of estrogen showing some response with methyltestosterone (K i=2.1×10 −5 M). On the other hand, the receptor assay is able to screen for estrogenic chemicals such as tamoxifen (K i=4.5×10 −9 M) and diethylstilbesterol (K i=8.1×10 −7 M). Therefore, E-6-F tracer is useful as a tracer for FPBA that is able to screen for chemicals structurally similar to estrogen using antibody, and that is able to screen for chemicals functionally similar to estrogen using receptor binding assay.