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Related Experiment Videos

Complement C5a receptor assay for high throughput screening.

S R Harris1, R K Garlick, J J Miller

  • 1E.I.Du Pont de Nemours, Biotechnology Systems Division, Billerica, MA 01862.

Journal of Receptor Research
|January 11, 1991
PubMed
Summary
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Researchers developed a stable C5a receptor assay using U937 cells and radiolabeled C5a. This reliable drug screening assay enables the discovery of novel compounds targeting the complement C5a receptor.

Area of Science:

  • Immunology
  • Pharmacology
  • Cell Biology

Background:

  • The complement C5a receptor plays a crucial role in inflammatory responses.
  • U937 cells, a human histiocytic lymphoma line, can be stimulated to express functional C5a receptors.

Purpose of the Study:

  • To develop a stable and reproducible drug screening assay for compounds targeting the complement C5a receptor.
  • To characterize the binding properties of the C5a receptor on U937 cells.

Main Methods:

  • Stabilization of complement C5a receptor on U937 cells.
  • Preparation of radiolabeled C5a ([125I]-Bolton Hunter) using reverse-phase HPLC.
  • Filtration binding assay for receptor saturation analysis.

Main Results:

Related Experiment Videos

  • The C5a receptor on U937 cells remained stable for over 3 months in a ready-to-use concentration.
  • Binding affinity (Kd) was determined to be 10-40 pM, with 50,000-100,000 receptor sites per cell.
  • A reliable and reproducible drug screening assay in kit format was successfully developed.

Conclusions:

  • The developed reagents and assay provide a convenient tool for screening compounds that modulate C5a receptor activity.
  • This assay facilitates the identification of potential therapeutics for complement-mediated inflammatory diseases.