title: A high-throughput homogeneous assay for reverse transcriptase using generic reagents and time-resolved fluorescence detection.
abstract: A homogeneous time-resolved fluorescence (HTRF) assay has been developed for determining the activity of HIV reverse transcriptase (HIV-RT). By using a sequential capping strategy, the assay has been configured to utilize only generic reagents such as biotinylated dUTP, streptavidin-allophycocyanin, and streptavidin-europium. The assay was optimized for a HIV-RT high-throughput screen. Under optimized conditions, a signal-to-background ratio of approximately 10:1 and a Z' factor of 0.8 were obtained. The titration curves of several known HIV-RT inhibitors have been evaluated with this assay format. This HTRF format can be used for high-throughput assays with other nucleotide polymerases.
authors.affiliation: DuPont Pharmaceuticals Research Laboratories, Wilmington, Delaware 19880-0400, USA. ji-hu.zhang@dupontpharma.com
authors.forename: J H
authors.initials: JH
authors.lastname: Zhang
authors.forename: T
authors.initials: T
authors.lastname: Chen
authors.forename: S H
authors.initials: SH
authors.lastname: Nguyen
authors.forename: K R
authors.initials: KR
authors.lastname: Oldenburg
mesh_terms: D006678:HIV / Q000201:enzymology*; D007202:Indicators and Reagents / Q000737:chemistry*; D012194:RNA-Directed DNA Polymerase / Q000032:analysis*; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods*
chemical_list: D007202:Indicators and Reagents; D012194:RNA-Directed DNA Polymerase