Determination of the relative potency of an anti-TNF monoclonal antibody (mAb) by neutralizing TNF using an In Vitro bioanalytical method

This protocol shows the measurement of the apoptotic activity neutralization of TNFα in a mouse fibroblast cell model (WEHI 164) using an anti-TNFα mAb. In addition, this protocol can be used to evaluate other anti-TNFα molecules, such as fusion proteins. The cellular model employed here is sensitive to TNFα-mediated apoptosis when an additional stress factor is induced in cell culture conditions (e.g., serum deprivation). This procedure exemplifies how to execute this analytical assay, highlighting the key operations relating to the sample preparation, cell dilution, apoptosis induction, and spectrophotometric measurements that are critical to ensure successful results. This protocol reveals the best-performance conditions relating to apoptosis induction and efficient signal recording, leading to low uncertainty values.