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.