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Related Experiment Videos

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

J H Zhang1, T Chen, S H Nguyen

  • 1DuPont Pharmaceuticals Research Laboratories, Wilmington, Delaware 19880-0400, USA. ji-hu.zhang@dupontpharma.com

Analytical Biochemistry
|June 28, 2000
PubMed
Summary

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A new homogeneous time-resolved fluorescence (HTRF) assay enables high-throughput screening of HIV reverse transcriptase (HIV-RT) activity using generic reagents. This optimized assay demonstrates robust performance for identifying potential HIV-RT inhibitors.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Drug Discovery

Background:

  • HIV reverse transcriptase (HIV-RT) is a critical target for antiviral therapies.
  • Developing efficient assays for HIV-RT activity is essential for identifying novel inhibitors.
  • Existing assays may require specialized reagents or lack scalability for high-throughput screening.

Purpose of the Study:

  • To develop and optimize a homogeneous time-resolved fluorescence (HTRF) assay for determining HIV-RT activity.
  • To configure the assay using generic reagents for broader applicability and cost-effectiveness.
  • To validate the assay's performance for high-throughput screening (HTS) and inhibitor evaluation.

Main Methods:

  • Development of a sequential capping strategy for the HTRF assay.

Related Experiment Videos

  • Utilization of generic reagents: biotinylated dUTP, streptavidin-allophycocyanin, and streptavidin-europium.
  • Optimization of the assay for HIV-RT high-throughput screening.

    • Main Results:

    • Achieved a signal-to-background ratio of approximately 10:1 under optimized conditions.
    • Obtained a Z' factor of 0.8, indicating excellent assay performance for HTS.
    • Successfully evaluated titration curves of known HIV-RT inhibitors using the developed HTRF format.

    Conclusions:

    • The developed HTRF assay is suitable for high-throughput screening of HIV-RT activity.
    • The assay's configuration with generic reagents makes it adaptable for other nucleotide polymerases.
    • This assay provides a robust platform for the discovery of new HIV-RT inhibitors.