A non-radioactive complement-dependent cytotoxicity assay for anti-CD20 monoclonal antibody

Area of Science:

• Immunology
• Biotechnology
• Analytical Chemistry

Background:

• Complement-dependent cytotoxicity (CDC) assays are crucial for evaluating monoclonal antibodies (mAbs).
• Existing methods for assessing anti-CD20 mAb potency can be complex or involve radioactivity.
• There is a need for simple, robust, and non-radioactive assays for mAb quality control.

Purpose of the Study:

• To develop and validate a simple, non-radioactive complement-dependent cytotoxicity (CDC) assay.
• To determine the relative potency of the anti-CD20 mAb IDEC-C2B8.
• To establish a high-throughput method for quality control of anti-CD20 mAbs.

Main Methods:

• Utilized the redox dye Alamar blue to measure viable cell numbers.
• Developed a 96-well microtiter plate-based assay for high-throughput screening.
• Established dose-response curves and assessed assay precision, accuracy, and specificity.

Main Results:

• Demonstrated a linear relationship between Alamar blue fluorescence and viable cell count.
• Achieved reproducible dose-response curves within the 0.02-3.3 µg/ml concentration range.
• Exhibited high precision (intra-assay: 5-12%, inter-assay: 6-10%) and accuracy (spike recoveries: 101-109%).
• Confirmed assay specificity and its ability to detect antibody degradation.

Conclusions:

• The Alamar blue-based CDC assay is a simple, accurate, and reproducible method for determining anti-CD20 mAb potency.
• The assay is suitable for high-throughput screening and stability-indicating quality control testing.
• This non-radioactive approach offers a favorable alternative to conventional cell viability assays.