A high-throughput homogeneous assay for reverse transcriptase using generic reagents and time-resolved fluorescence detection.
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.
Zhang
J H
JH
DuPont Pharmaceuticals Research Laboratories, Wilmington, Delaware 19880-0400, USA. ji-hu.zhang@dupontpharma.com
Chen
T
T
Nguyen
S H
SH
Oldenburg
K R
KR
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*
D007202:Indicators and Reagents; D012194:RNA-Directed DNA Polymerase