Reversed-phase liquid chromatography coupled on-line to estrogen receptor bioaffinity detection based on fluorescence polarization |
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Authors: | Jelle Reinen Jeroen Kool Nico P E Vermeulen |
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Institution: | (1) Department of Chemistry and Pharmaceutical Sciences, LACDR-Division of Molecular Toxicology, Vrije Universiteit, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands;(2) Biomolecular Analysis, Department of Chemistry and Pharmaceutical Sciences, Vrije Universiteit, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands |
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Abstract: | We describe the development and validation of a high-resolution screening (HRS) platform which couples gradient reversed-phase
high-performance liquid chromatography (RP-HPLC) on-line to estrogen receptor α (ERα) affinity detection using fluorescence
polarization (FP). FP, which allows detection at high wavelengths, limits the occurrence of interference from the autofluorescence
of test compounds in the bioassay. A fluorescein-labeled estradiol derivative (E2-F) was synthesized and a binding assay was
optimized in platereader format. After subsequent optimization in flow-injection analysis (FIA) mode, the optimized parameters
were translated to the on-line HRS bioassay. Proof of principle was demonstrated by separating a mixture of five compounds
known to be estrogenic (17β-estradiol, 17α-ethinylestradiol and the phytoestrogens coumestrol, coumarol and zearalenone),
followed by post-column bioaffinity screening of the individual affinities for ERα. Using the HRS-based FP setup, we were
able to screen affinities of off-line-generated metabolites of zearalenone for ERα. It is concluded that the on-line FP-based
bioassay can be used to screen for the affinity of compounds without the disturbing occurrence of autofluorescence. |
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Keywords: | Fluorescence polarization High-resolution screening (HRS) Estrogen receptor α Phytoestrogens On-line bioaffinity assay Receptor affinity detection (RAD) |
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