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Human epidermal growth factor receptor 2 (HER2) is an important biomarker for detection and treatment of breast cancer. In this study, we developed monoclonal antibodies against the extracellular domain (ECD) of HER2 and established a rapid and accurate lateral flow immunoassay (LFIA) for use in community medical institutions. The gene sequence of human HER2-ECD was obtained from the National Center for Biotechnology Information (NCBI) to construct the expression plasmid. HER2-ECD protein expressed in HEK293F cells was used to immunize BALB/c mice. The monoclonal antibodies were produced in mouse ascites and isolated by hybridoma cell screening. Antibodies were analyzed for purity by SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel-electrophoresis) and affinity was assessed by enzyme-linked immunosorbent assay (ELISA) while subtypes were detected using the commercial kits. The HER2-ECD test strip was prepared based on the sandwich method and evaluated using a portable detection instrument. The affinity of the paired antibodies, 4D8 and 8D9, both reached 1 × 108 L/mol. Both antibodies specifically recognized the HER2-ECD protein in serum. The limit of detection (LOD) of the gold nanoparticle (AuNP)-based LFIA was 1.7 ng/mL with a detection range of 1.7–400 ng/mL, and the performance of the HER2-ECD strip correlated well with that of a Siemens chemiluminescent immunoassay (CLIA) kit. In conclusion, the paired antibodies were successfully prepared with high affinity and specificity. The AuNP-based LFIA of HER2-ECD provides a fast and accurate method to detect the concentration of HER2-ECD in serum samples for clinical use in community medical institutions, and could contribute to determining the progress of the disease or the effectiveness of treatment.
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