Detection of anti-drug antibodies to trastuzumab in serum by enzyme-linked immunosorbent assay

INTRODUCTION . Trastuzumab is a recombinant humanised monoclonal antibody against human epidermal growth factor receptor 2 (HER2) approved as targeted therapy for patients with HER2-positive breast cancer. Improving overall patient survival, trastuzumab has become a standard of care in cancer treatment. However, trastuzumab can induce anti-drug antibodies (ADAs), which can reduce the effectiveness of treatment. Therefore, timely detection of serum ADAs is necessary for potential treatment adjustment. AIM . This study aimed to develop and validate an enzyme-linked immunosorbent assay (ELISA) for semiquantitative determination of all-class anti-trastuzumab ADAs. MATERIALS AND METHODS . The presented test system is a classic ELISA kit comprising a 96-well plate with trastuzumab immobilised on the inner surface of the wells, a solution of secondary antibody (trastuzumab conjugated with horseradish peroxidase), a substrate solution (3,3’,5,5’-tetramethylbenzidine), and a stop solution. The authors determined the optical density at 450 nm using a microplate photometer. Quality control solutions were prepared by diluting monoclonal antibodies against trastuzumab with a buffer containing human serum. RESULTS . The limit of quantification for ADAs is 2 ng/mL, and the minimum required dilution is 1:2. The assay is tolerant to trastuzumab concentrations of 116 ng/mL and 1000 ng/mL and can detect ADA levels of 16 ng/mL and 100 ng/mL, respectively, in the presence of trastuzumab. The analytical procedure provides results with acceptable specificity, selectivity, and within-run and between-run precision. The assay can measure ADA concentrations ranging from 640 ng/mL to 0.16 ng/mL without exhibiting the hook effect from excess ADA levels. ADAs are stable for 6 hours at room temperature, 90 days at −70 °C, and three freeze–thaw cycles with 12-hour periods at −70 °C. CONCLUSIONS . The test system parameters established in this study confirm the applicability of the system for detecting ADAs against trastuzumab in biological fluids during targeted therapy.