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A novel high-through-put assay for screening of pro-apoptotic drugs
Maria Hägg1, Kenneth Bivén, Takayuki Ueno
1Cancer Center Karolinska, Department of Oncology and Pathology, Karolinska Institute and Hospital, Stockholm, Sweden.
Abstract:
Screening for anti-cancer substances is commonly conducted using viability assays. An inherent problem with this approach is that all compounds that are toxic and growth inhibitory, irrespective of mechanism of action, will score positive. It would be beneficial to be able to screen for compounds that specifically induce apoptosis. We here describe an ELISA-assay based on a monoclonal antibody (M30) which recognizes a neo-epitope on cytokeratin 18 exposed after cleavage by caspases during apoptosis. We show that this assay detects apoptosis in epithelial cells and that the sensitivity is sufficient for screening in the 96-well format. We used the M30-ELISA assay to screen 500 low molecular weight compounds from a chemical library from the National Cancer Institute and identified 16 drugs with strong pro-apoptotic activity, suggesting that the assay is a useful tool for discovery of pro-apoptotic drugs.
Insights
This study introduces a new ELISA assay using M30 antibody to detect apoptosis. The assay successfully identified 16 pro-apoptotic drugs from a chemical library, offering a specific method for anti-cancer drug discovery.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Research
Background:
- Viability assays are standard for anti-cancer drug screening but lack specificity, identifying all toxic compounds.
- Distinguishing compounds that induce apoptosis is crucial for targeted anti-cancer drug development.
- There is a need for specific assays to screen for pro-apoptotic agents.
Purpose of the Study:
- To develop and validate an ELISA-based assay for specifically detecting apoptosis.
- To utilize this assay for screening chemical libraries to identify pro-apoptotic compounds.
- To establish a sensitive and efficient tool for discovering novel anti-cancer drugs.
Main Methods:
- Development of an Enzyme-Linked Immunosorbent Assay (ELISA) utilizing the M30 monoclonal antibody.
- The M30 antibody recognizes a neo-epitope on cytokeratin 18, exposed during caspase-mediated apoptosis.
- Assay validation in epithelial cells and application to screen 500 low molecular weight compounds.
Main Results:
- The M30-ELISA assay demonstrated reliable detection of apoptosis in epithelial cells.
- The assay exhibited sufficient sensitivity for screening in a 96-well format.
- Screening identified 16 compounds with significant pro-apoptotic activity from the National Cancer Institute library.
Conclusions:
- The M30-ELISA assay is a valuable tool for specifically detecting and screening for pro-apoptotic substances.
- This assay facilitates the discovery of novel anti-cancer drugs with a targeted mechanism of action.
- The findings support the utility of apoptosis-specific assays in anti-cancer drug discovery pipelines.