Validated Immunochemical Assay for Comprehensive Determination of the Human Epidermal Growth Factor Receptor 2 Released from and Bound to Cells

Human epidermal growth factor receptor 2 (HER2) is a well-established cancer marker. It became a very successful diagnostic and therapeutic target, especially in breast cancer and other HER2-expressing cancer types. In the clinic, the gold-standard immunohistochemical diagnostic methods employing the specific anti-HER2 antibodies are used to measure the expression level of the membrane-bound receptor. The soluble extracellular domain (ECD) of HER2 that is released from the overexpressing cells circulates in the blood and can reflect the tissue expression of the receptor. There is a need for accurate and validated assays to correlate the concentration of the circulating HER2 protein with disease clinical manifestations. Our team has developed and validated the novel sandwich enzyme-linked immunosorbent assay (ELISA) for quantification of the membrane-bound and the released from cells ECD domain of HER2. The assay uses two unique monoclonal antibodies specific to HER2 developed previously. The quantitation range includes HER2 concentration from 1.56-100 ng/mL, which is expected for cancer cells cultured in vitro and shows sensitivity at the level of 0.5 ng/mL. The satisfactory intra- and inter-assay precision and accuracy of the method make it applicable for HER2 quantification in various types of biological samples, including cell culture medium, serum, and solid tumor tissue. Here, we focus on the comprehensive determination of the receptor-associated and secreted by the in vitro cultured cancer cells. The paper presents a step-by-step protocol for the quantification of HER2 protein that can be employed for testing a variety of cell lines, blood, and tissues.