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Related Experiment Videos

A generic particle-based nonradioactive homogeneous multiplex method for high-throughput screening using microvolume

C Martens1, A Bakker, A Rodriguez

  • 1Affymax Research Institute, 4001 Miranda Avenue, Palo Alto, California 94304, USA.

Analytical Biochemistry
|August 24, 1999
PubMed
Summary

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We developed a novel fluorescence assay for high-throughput screening. This assay is homogeneous, miniaturizable, and multiplex-capable, showing promise for drug discovery.

Area of Science:

  • Biochemistry
  • Assay Development
  • Drug Discovery

Background:

  • High-throughput screening (HTS) is crucial for identifying novel drug candidates.
  • Existing screening methods can be limited by radioactivity, complexity, or lack of multiplexing capability.

Purpose of the Study:

  • To develop a novel, homogeneous, fluorescence-based binding assay for high-throughput screening.
  • To validate the assay's performance for receptor-ligand binding.
  • To demonstrate the assay's capability for multiplexing and miniaturization.

Main Methods:

  • A fluorescence-based homogeneous binding assay utilizing Cy5- or Cy5.5-labeled ligands and immobilized receptors on particles (beads or cells).
  • Detection of localized fluorescence using a modified microvolume fluorimeter (MVF).

Related Experiment Videos

  • Image processing software for event enumeration and fluorescence intensity analysis.
  • Development of a multiplex assay by immobilizing different-sized beads with distinct receptors (IL-1R and IL-5R).

    • Main Results:

    • The MVF assay accurately determined IC(50) values for IL-1 receptor ligands, consistent with radioligand binding assays.
    • A multiplex assay successfully quantified ligands for IL-1R and IL-5R simultaneously with comparable IC(50) values.
    • The assay demonstrated reliable performance in a miniaturized 16-microl volume within an 864-well plate format.

    Conclusions:

    • The developed homogeneous, nonradioactive, miniaturizable, and multiplex-capable fluorescence assay is highly promising for screening compound libraries.
    • This novel assay offers an efficient and versatile platform for drug discovery and development.
    • The assay's ability to perform multiplexed and miniaturized screening enhances its utility for large-scale compound collections.