Development and Validation of the ELISA Method for Anti-trastuzumab Antibodies Determination in Human Serum

Introduction. One of the widely used specific anti-HER2 (human epidermal growth factor receptor 2) MAb drugs is trastuzumab. Trastuzumab is highly effective for malignant HER2 hyperexpression reduction, which results in HER2 oncogenicity decrease. As any other biotherapeutics trastuzumab can cause immunological adverse reactions, e.g. immunogenicity or anti-drug antibodies (ADAs) production.Aim. The aim of this study was to develop and validate the analytical method for anti-trastuzumab antibodies determination in human blood serum.Materials and methods. The semi-quantitative anti-trastuzumab antibody determination was carried out by the ELISA method combined with ACE technique, using spectrophotometric detection in the visible range of the spectrum.Results and discussion. The developed method was validated for cut point, selectivity, sensitivity, "hook" effect, drug tolerance, precision and stability (short-term and long-term). To decrease the background noise from non-specific binding of sera components, the minimum required dilution value was determined at 10 % serum. The calculated values for screening cut point (normalization factor) and confirmatory cut point were 0.004 and 34.59 %, respectively. The sensitivity of the developed method was estimated at 99.5 ng/mL of anti-trastuzumab antibodies.Conclusion. The obtained results allow us to use the developed ACE ELISA method for the determination of anti-trastuzumab antibodies in human serum during trastuzumab safety clinical trials.