An improved assay for antibody dependent cellular cytotoxicity based on time resolved fluorometry

Area of Science:

• Immunology
• Cellular Cytotoxicity Assays
• Biochemistry

Background:

• Traditional assays for antibody-dependent cellular cytotoxicity (ADCC) often rely on the release of chromium-51 (51Cr), a radioisotope with associated handling and disposal concerns.
• There is a need for safer, faster, and equally effective methods to assess ADCC, crucial for monitoring therapeutic antibody efficacy and production.
• Wein 133 cells, a B cell non-Hodgkin's lymphoma cell line expressing CDw52, are suitable targets for ADCC studies.

Purpose of the Study:

• To describe and validate a new, non-hazardous assay for quantifying antibody-dependent cellular cytotoxicity (ADCC) based on europium (EuDTPA) release.
• To compare the performance of the novel europium release assay with the established chromium-51 (51Cr) release assay for ADCC measurement.
• To highlight the advantages of the europium release assay for routine laboratory applications, including monitoring monoclonal antibody production.

Main Methods:

• Target Wein 133 cells were labeled with europium diethylenetriaminopentaacetic acid (EuDTPA).
• Cells were sensitized with the anti-lymphocytic monoclonal antibody Campath-1H and incubated with human peripheral blood mononuclear cells to induce ADCC.
• Europium release was quantified by measuring fluorescence of the EuDTPA chelate after addition of enhancement solution (2-naphthoyl trifluoroacetone, 2-NTA) using time-resolved fluorometry.

Main Results:

• The novel EuDTPA release assay demonstrated comparable results to traditional 51Cr release assays for measuring ADCC.
• The assay proved to be non-hazardous and has been successfully used for over two years in routine monitoring of Campath-1H production and purification.
• The method allows for sensitive detection of cell lysis through time-resolved fluorometry of the fluorescent chelate.

Conclusions:

• The described europium (EuDTPA) release assay is a viable, safer, and faster alternative to radioisotope-based methods for ADCC determination.
• This assay is suitable for widespread adoption in laboratories performing ADCC assays, offering significant advantages in terms of safety and practicality.
• The assay's reliability and ease of use make it an effective tool for quality control in the production of therapeutic antibodies like Campath-1H.