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Updated: Dec 29, 2025
Cell-based Flow Cytometry Assay to Measure Cytotoxic Activity
Published on: December 17, 2013
Flow cytometry-based assessment of direct-targeting anti-cancer antibody immune effector functions
Michelle L Miller1, Olivera J Finn1
1Department of Immunology, University of Pittsburgh School of Medicine, Pittsburgh, PA, United States.
Abstract:
Monoclonal antibody-based therapies are increasingly being used to treat cancer. Some mediate their therapeutic effects through modifying the function of immune cells globally, while others bind directly to tumor cells and can recruit immune effector cells through their Fc regions. As new direct-binding agents are developed, having the ability to test their Fc-mediated functions in a high-throughput manner is important for selecting antibodies with immune effector properties. Here, using monoclonal anti-CD20 antibody (rituximab) as an example and the CD20+ Raji cell line as tumor target, we describe flow cytometry-based assays for determining an antibody's capacity for mediating antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and complement-dependent cytotoxicity (CDC). These assays are sensitive, reliable, affordable and avoid the use of radioactivity.
Insights
New flow cytometry assays enable high-throughput testing of monoclonal antibody functions. These methods assess antibody-dependent cellular cytotoxicity (ADCC), phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC) without radioactivity.
Area of Science:
- Immunology
- Oncology
- Biotechnology
Background:
- Monoclonal antibodies are key cancer therapies, acting via immune cell modulation or direct tumor cell binding.
- Fc-mediated effector functions are crucial for direct-binding antibodies, necessitating robust testing methods.
- High-throughput assays are needed to evaluate Fc-mediated functions for selecting effective antibodies.
Purpose of the Study:
- To develop and validate flow cytometry-based assays for assessing Fc-mediated functions of monoclonal antibodies.
- To evaluate antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC).
Main Methods:
- Utilized flow cytometry to measure ADCC, ADCP, and CDC.
- Employed monoclonal anti-CD20 antibody (rituximab) and CD20+ Raji cells as a model system.
- Developed assays that are sensitive, reliable, affordable, and do not require radioactivity.
Main Results:
- Demonstrated the capacity of flow cytometry assays to quantify ADCC, ADCP, and CDC.
- Validated the utility of these assays using a well-characterized antibody-target system.
- Confirmed the assays' sensitivity, reliability, and cost-effectiveness.
Conclusions:
- Flow cytometry assays provide a powerful tool for high-throughput screening of antibody Fc-mediated effector functions.
- These assays facilitate the selection of monoclonal antibodies with optimal immune effector properties for cancer therapy.
- The developed methods offer a practical and efficient alternative to traditional, radioactivity-based assays.
