Establishment of a reporter gene-based assay to determine ADCC activity of anti-tetanus toxin monoclonal antibody

Area of Science:

• Immunology
• Biotechnology
• Cell Biology

Background:

• Tetanus remains a significant public health issue.
• Effective therapeutic monoclonal antibodies targeting the tetanus toxin C-fragment (TT-C) are under development.
• Reliable assays are needed to assess the antibody-dependent cell-mediated cytotoxicity (ADCC) activity of these antibodies.

Purpose of the Study:

• To develop and validate a robust luciferase reporter ADCC assay.
• To utilize a stable cell line expressing high levels of TT-C as target cells.
• To provide a reliable platform for the functional characterization and quality control of anti-tetanus toxin neutralizing monoclonal antibodies.

Main Methods:

• Development of a stable Flp-In CHO cell line expressing high levels of TT-C.
• Establishment and validation of a luciferase reporter ADCC assay using these TT-C target cells.
• Method validation including specificity, precision, and accuracy assessment.

Main Results:

• The TT-C expressing CHO cell line demonstrated stable and high expression (99.36% TT-C-positive).
• The assay generated a highly reproducible sigmoidal dose-response curve (R² > 0.99).
• Method validation confirmed high specificity, precision (RSD = 13.90%), and accuracy (99.99%-103.18% recovery).

Conclusions:

• A validated luciferase reporter ADCC assay has been successfully developed.
• This assay provides a reliable platform for potency evaluation and quality control of anti-tetanus antibodies.
• The assay supports the development of effective tetanus toxin neutralizing monoclonal antibodies.