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Related Experiment Videos

A Fluorescence-Based High Throughput Screen for the Transporter Associated with Antigen Processing.

Blevitt1, Früh, Glass

  • 1R.W. Johnson Pharmaceutical Research Institute, San Diego, California.

Journal of Biomolecular Screening
|June 6, 2000
PubMed
Summary
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A new nonradioactive assay rapidly measures transporter associated with antigen processing (TAP) peptide transport activity. This high-throughput screening (HTS) method uses fluorescent labels and filtration plates for efficient drug discovery.

Area of Science:

  • Immunology
  • Biochemistry
  • Drug Discovery

Background:

  • Transporter associated with antigen processing (TAP) is crucial for MHC class I antigen presentation.
  • Traditional TAP assays use radioactive peptides and are time-consuming, limiting throughput.
  • Drug discovery necessitates rapid, reliable screening of numerous samples for bioactivity.

Purpose of the Study:

  • To develop a nonradioactive, high-throughput screening (HTS) assay for evaluating TAP peptide transport activity.
  • To replace radioactive labeling with fluorescent labeling without sacrificing assay sensitivity or peptide transport efficiency.
  • To adapt the HTS assay for automated platforms to enable large-scale compound screening.

Main Methods:

  • Developed a nonradioactive assay utilizing fluorescently labeled peptides to measure TAP transport.

Related Experiment Videos

  • Employed multiscreen filtration plates to increase throughput and eliminate centrifugation steps.
  • Optimized assay parameters including time course, dose response, signal/noise ratio, and reagent stability on a 96-channel pipetting station.

    • Main Results:

    • The nonradioactive HTS assay demonstrated comparable kinetic characteristics to traditional methods.
    • The assay successfully replaced radioactive labels with fluorescent labels, maintaining sensitivity and transport efficiency.
    • The HTS system was optimized for automated screening, achieving a throughput of up to 17,600 compounds per week.

    Conclusions:

    • A novel, nonradioactive HTS assay for TAP peptide transport activity has been successfully developed and validated.
    • This HTS assay significantly enhances efficiency and throughput for evaluating TAP function in drug discovery.
    • The developed system facilitates rapid screening of compound libraries for potential therapeutic agents targeting antigen presentation pathways.