Document Detail

Assessment of a robust model protocol with accelerated throughput for a human recombinant full length estrogen receptor-alpha binding assay: protocol optimization and intralaboratory assay performance as initial steps towards validation.
MedLine Citation:
PMID:  20074635     Owner:  NLM     Status:  MEDLINE    
Despite about two decades of research in the field of endocrine active compounds, still no validated human recombinant (hr) estrogen receptor-alpha (ERalpha) binding assay is available, although hr-ERalpha 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-ERalpha and performance in a 96-well plate format. A full length hr-ERalpha 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 [17beta-estradiol, norethynodrel, dibutylphthalate] nine test compounds with different affinities for the ERalpha [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 ERalpha 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 ERalpha ligand binding domain. Irrespective of the chemical nature of the compound, individual IC(50)-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 ERalpha 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.
Alexius Freyberger; Vickie Wilson; Marc Weimer; Shirlee Tan; Hoai-Son Tran; Hans-Jürgen Ahr
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2010-01-13
Journal Detail:
Title:  Reproductive toxicology (Elmsford, N.Y.)     Volume:  30     ISSN:  1873-1708     ISO Abbreviation:  Reprod. Toxicol.     Publication Date:  2010 Aug 
Date Detail:
Created Date:  2010-07-15     Completed Date:  2010-11-09     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  8803591     Medline TA:  Reprod Toxicol     Country:  United States    
Other Details:
Languages:  eng     Pagination:  50-9     Citation Subset:  IM    
Copyright Information:
Copyright 2010 Elsevier Inc. All rights reserved.
Bayer Schering Pharma AG, BSP-GDD-GED-GTOX Special Toxicology, Wuppertal, Germany.
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MeSH Terms
Animal Testing Alternatives*
Binding, Competitive
Biological Assay / methods*,  standards
Dose-Response Relationship, Drug
Endocrine Disruptors / pharmacology*
Estrogen Receptor alpha / chemistry,  metabolism*
Models, Biological*
Protein Binding
Radioligand Assay
Recombinant Proteins / chemistry,  metabolism*
Reproducibility of Results
Reg. No./Substance:
0/Endocrine Disruptors; 0/Estrogen Receptor alpha; 0/Ligands; 0/Recombinant Proteins

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine

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