A High-Throughput Homogeneous Assay for Reverse Transcriptase Using Generic Reagents and Time-Resolved Fluorescence Detection

✍ Scribed by Ji-Hu Zhang; Tongming Chen; Son H. Nguyen; Kevin R. Oldenburg

• Publisher: Elsevier Science
• Year: 2000
• Tongue: English
• Weight: 72 KB
• Volume: 281
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.2000.4567

✦ Synopsis

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.