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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
Summary
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:
- 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.