High throughput adjustable 96-well plate assay for androgen receptor binding: a practical approach for EDC screening using the chimpanzee AR.
The issue as to whether natural and man-made chemicals interfere with endocrine function has raised concerns. This interference could be biologically significant even at very low doses if the chemicals interact deleteriously with hormone receptors at low concentrations. Therefore, the United States Environmental Protection Agency (USEPA) Office of Coordination and Policy (OSCP) requested that a nonhuman mammalian androgen receptor binding assay be developed for possible use in their Endocrine Disruptor Screening Program (EDSP). Ideally, this assay would be high throughput, not use animals as a source of receptor protein, easily deployed throughout the scientific community, utilize reagents available to both the public and private sector, and have the potential for future automation. We developed a highly modified 96-well plate assay which meets these criteria. It employs a baculovirus expressed recombinant primate androgen receptor which is publically available and exploits the unique ability of some mammalian androgen receptors to remain biologically active after guanidine hydrochloride (GdnHCl) solubilization. This GdnHCl treated receptor remains soluble and requires no additional purification prior to use. We provide a very detailed description of the assay protocol itself, and similarly detailed method for producing and solubilizing the receptor.
Hartig
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Reproductive Toxicology Division, 2525 East Highway 54, MD 72, USEPA, ORD, NHEERL, RTP, NC 27711, USA. hartig.Phillip@epa.gov
Cardon
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Blystone
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Gray
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Wilson
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D000818:Animals; D001667:Binding, Competitive; D002460:Cell Line; D052244:Endocrine Disruptors / Q000378:metabolism*; D019791:Guanidine / Q000737:chemistry; D002679:Pan troglodytes; D051381:Rats; D011944:Receptors, Androgen / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D018411:Spodoptera
C410798:AR protein, human; D052244:Endocrine Disruptors; D011944:Receptors, Androgen; D011994:Recombinant Proteins; D019791:Guanidine