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Related Experiment Videos

A multiwell format assay for heparanase.

Farhad Behzad1, Paul E C Brenchley

  • 1Department of Medicine, University of Manchester, M13 9WL Manchester, UK. farhad.behzad@talk21.com

Analytical Biochemistry
|August 21, 2003
PubMed
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This study introduces a novel assay for detecting heparanase activity using a biotinylated heparan sulfate glycosaminoglycan (HSGAG) substrate. The assay offers improved sensitivity, specificity, and ease of use for analyzing heparanase in various samples.

Area of Science:

  • Biochemistry
  • Enzymology
  • Assay Development

Background:

  • Heparanase is a key enzyme involved in various physiological and pathological processes.
  • Existing assays for heparanase activity often lack sensitivity, specificity, or ease of use.
  • A robust and reliable method for quantifying heparanase activity is needed for research and clinical applications.

Purpose of the Study:

  • To develop and validate a novel, sensitive, and specific assay for detecting heparanase activity.
  • To enable high-throughput screening of heparanase inhibitors.
  • To facilitate the measurement of heparanase levels in biological samples.

Main Methods:

  • A biotinylated heparan sulfate glycosaminoglycan (HSGAG) substrate was covalently linked to 96-well immunoassay plates.

Related Experiment Videos

  • Optimized incubation conditions allowed for the digestion of HSGAG by heparanase.
  • Loss of biotin signal, detected via peroxidase-streptavidin and colorimetric substrate, quantified heparanase activity.

    • Main Results:

    • The assay demonstrated high specificity, with no significant degradation observed by other tested enzymes.
    • The assay showed improved sensitivity and ease of handling compared to existing methods.
    • The covalent linkage of the substrate prevented leaching and allowed for long-term storage of coated plates.

    Conclusions:

    • A novel, sensitive, and specific assay for heparanase activity has been developed.
    • This assay is suitable for analyzing heparanase in clinical samples and cell culture supernatants.
    • The assay is ideal for screening potential heparanase antagonists.