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.

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