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A high-throughput homogeneous assay for reverse transcriptase using generic reagents and time-resolved fluorescence detection.

J H Zhang·T Chen·S H Nguyen·K R Oldenburg
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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.

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HIVIndicators and ReagentsRNA-Directed DNA PolymeraseSensitivity and SpecificitySpectrometry, FluorescenceJournal Article

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J H Zhang, T Chen, S H Nguyen, K R Oldenburg. 2000-06-01. A high-throughput homogeneous assay for reverse transcriptase using generic reagents and time-resolved fluorescence detection.. https://doi.org/10.1006/abio.2000.4567

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