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A very sensitive coupled luminescent assay for cytotoxicity and complement-mediated lysis
M J Corey1, R J Kinders, L G Brown
1University of Washington Medical School, Seattle 98195, USA.
Journal of Immunological Methods
|August 22, 1997
Summary
A new luminometric assay accurately measures cell death by detecting released glyceraldehyde-3-phosphate dehydrogenase (G3PDH). This sensitive assay enhances complement-mediated killing detection for cancer research.
Area of Science:
- Biochemistry
- Immunology
- Cell Biology
Background:
- Complement Factor H is a key regulator of complement pathways and a newly identified tumor-associated antigen.
- Increased demand exists for sensitive methods to measure cytotoxicity and complement-mediated killing.
- Tumor-associated antigens like Complement Factor H present therapeutic targets.
Purpose of the Study:
- To develop a simple, sensitive luminometric assay for measuring cell death.
- To quantify the release of glyceraldehyde-3-phosphate dehydrogenase (G3PDH) from damaged cells.
- To assess the assay's utility in detecting antibody-enhanced complement-mediated killing.
Main Methods:
- A coupled enzymatic assay was designed to measure G3PDH activity.
- G3PDH activity was linked to ATP production, measured via firefly luciferase.
- The assay detects G3PDH release from approximately 0.03 nucleated human cells or 1 rabbit erythrocyte.
Main Results:
- The assay demonstrated high sensitivity in detecting cell death.
- The assay utilizes inexpensive, commercially available reagents.
- An anti-factor-H antibody significantly enhanced complement-mediated killing of Raji cancer cells (>1000%).
Conclusions:
- The developed luminometric assay provides a sensitive and convenient method for measuring cytotoxicity.
- This assay is valuable for studying complement-mediated killing, particularly in cancer research.
- The findings highlight the potential of targeting Complement Factor H for cancer therapy.