کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
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2594314 | 1132264 | 2010 | 10 صفحه PDF | دانلود رایگان |
![عکس صفحه اول مقاله: Assessment of a robust model protocol with accelerated throughput for a human recombinant full length estrogen receptor-α binding assay: Protocol optimization and intralaboratory assay performance as initial steps towards validation Assessment of a robust model protocol with accelerated throughput for a human recombinant full length estrogen receptor-α binding assay: Protocol optimization and intralaboratory assay performance as initial steps towards validation](/preview/png/2594314.png)
Despite about two decades of research in the field of endocrine active compounds, still no validated human recombinant (hr) estrogen receptor-α (ERα) binding assay is available, although hr-ERα is available from several sources. In a joint effort, US EPA and Bayer Schering Pharma with funding from the EU-sponsored 6th framework project, ReProTect, developed a model protocol for such a binding assay. Important features of this assay are the use of a full length hr-ERα and performance in a 96-well plate format. A full length hr-ERα was chosen, as it was considered to provide the most accurate and human-relevant results, whereas truncated receptors could perform differently. Besides three reference compounds [17β-estradiol, norethynodrel, dibutylphthalate] nine test compounds with different affinities for the ERα [diethylstilbestrol (DES), ethynylestradiol, meso-hexestrol, equol, genistein, o,p′-DDT, nonylphenol, n-butylparaben, and corticosterone] were used to explore the performance of the assay. Three independent experiments per compound were performed on different days, and dilutions of test compounds from deep-frozen stocks, solutions of radiolabeled ligand and receptor preparation were freshly prepared for each experiment. The ERα binding properties of reference and test compounds were well detected. As expected dibutylphthalate and corticosterone were non-binders in this assay. In terms of the relative ranking of binding affinities, there was good agreement with published data obtained from experiments using a human recombinant ERα ligand binding domain. Irrespective of the chemical nature of the compound, individual IC50-values for a given compound varied by not more than a factor of 2.5. Our data demonstrate that the assay was robust and reliably ranked compounds with strong, weak, and no affinity for the ERα with high accuracy. It avoids the manipulation and use of animals, i.e., the preparation of uterine cytosol as receptor source from ovariectomized rats, as a recombinant protein is used and thus contributes to the 3R concept (reduce, replace, and refine). Furthermore, in contrast to other assays, this assay could be adjusted to an intermediate/high throughput format. On the whole, this assay is a promising candidate for further validation.
Journal: Reproductive Toxicology - Volume 30, Issue 1, August 2010, Pages 50–59