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Related Experiment Videos

Quantitative cell-adhesion assay for Clostridium difficile cytotoxin.

B C Mayall1, C Edmonds, G E Griffin

  • 1Department of Communicable Diseases, St George's Hospital Medical School, Tooting, London.

Journal of Medical Microbiology
|November 1, 1987
PubMed
Summary

A new assay quantifies Clostridium difficile cytotoxin by measuring fibroblast cell adhesion inhibition. This method offers a rapid, reproducible, and quantitative approach for toxin detection.

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Area of Science:

  • Microbiology
  • Cell Biology
  • Biochemistry

Background:

  • Clostridium difficile is a significant cause of infectious diarrhea.
  • Accurate and rapid detection of C. difficile cytotoxin is crucial for patient management.
  • Existing methods for cytotoxin detection can be time-consuming or lack quantitative precision.

Purpose of the Study:

  • To develop a novel, quantitative assay for Clostridium difficile cytotoxin.
  • To establish a reproducible method for measuring cytotoxin activity based on cell adhesion inhibition.
  • To explore the potential for wider application of this assay in studying cell adhesion-disrupting agents.

Main Methods:

  • Utilized suspended fibroblasts (BHK cells) that fail to adhere to plastic when exposed to cytotoxin.

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  • Employed a dye-binding technique (Coomassie blue R-250) for spectrophotometric quantification of adherent cells, avoiding microscopy.
  • Purified the cytotoxin using gel-permeation and ion-exchange chromatography, confirming purity via SDS-PAGE.

    • Main Results:

    • The dye-binding assay demonstrated a direct proportionality between eluted dye and the number of adherent cells.
    • Cytotoxin exposure inhibited BHK cell adhesion in a dose-dependent manner, influenced by time and temperature.
    • The assay proved to be quantitative, rapid, and reproducible for C. difficile cytotoxin detection.

    Conclusions:

    • A robust and quantitative assay for C. difficile cytotoxin has been successfully developed.
    • The assay's principle, based on inhibition of cell adhesion, has potential applications for other toxins.
    • This method provides a valuable tool for C. difficile research and diagnostics.