Circulating HER2 extracellular domain (HER2 ECD) levels were proposed as a surrogate for HER2 tissue expression to monitor breast cancer patients for early relapse or responses to standard or HER2-targeted therapies, such as the monoclonal antibody (mAb) trastuzumab. Currently, available commercial ELISA assays for HER2 ECD rely on antibodies recognizing undisclosed or unknown epitopes. In this work, two ELISA assays employing MGR2 and MGR3 epitope-specific mAbs for HER2 ECD were developed and validated, showing good assay precision and linearity of the dose-response signal within the dynamic range of 0.19-12.50 ng mL −1 and detection limits of 0.76 and 0.75 ng mL −1 for the MGR2 and MGR3 assays, respectively. The developed assay showed a good agreement with two widely used commercial kits for HER2 ECD quantification in serum samples from breast cancer patients. A complete characterization of mAb-HER2 ECD interaction was performed by means of surface plasmon resonance using trastuzumab as control for both epitope mapping and kinetics analysis. The epitopes recognized by the two mAbs showed no overlap with trastuzumab, which was confirmed by trastuzumab interference analysis in serum samples. The method showed to be a practical approach to determine HER2 ECD with a high degree of sensitivity, reliability and recovery in samples containing mAbs-based therapies.HER2 is a trans-membrane tyrosine kinase receptor, member of the epidermal growth factor receptor (EGFR) family. Abnormal expression of HER2 was observed in a number of primary tumors, including breast cancer (BC) and gastric cancer, where HER2 gene is overexpressed in approximately 30% and 24% of patients, respectively 1,2 . Determination of HER2 overexpression in BC tissue has a role in clinical decisions, as it is associated with an unfavourable prognosis, especially in patients with lymph node metastases 3 , with greater resistance to hormonal therapy and some chemotherapeutical agents 4,5 and with increased sensitivity to other chemotherapy regimens 6,7 . Moreover, HER2 protein tissue expression has been employed as an important predictive factor to take decision about anti-HER2 therapeutic approaches such as the use of monoclonal antibodies (mAbs) trastuzumab (Herceptin ™ , Genentech) and pertuzumab (Perjeta ™ , Genentech) 8 . The combination of trastuzumab www.nature.com/scientificreports www.nature.com/scientificreports/ mAbs -HER2 ECD SPR kinetics experiments. Several data are available in literature concerning the affinity constant (K D ) determination for trastuzumab-HER2 ECD interaction using Biacore instruments. In general, all data refer to concentration ranging from sub-nanomolar to nanomolar.In particular, K D of 0.4 nM (k a = 4.9 × 10 5 M −1 s −1 ; k d = 1.7 × 10 −4 s −1 ) 26 , 1.9 nM (k a = 1.9 × 10 6 M −1 s −1 ; k d = 3.4 × 10 −3 s −1 ) 27 , 0.10 nM (k a = 0.34 × 10 6 M −1 s −1 ; k d = 0.42 × 10 −4 s −1 ) 28 , 0.49 nM (k a = 8.46 × 10 4 M −1 s −1 ; k d = 4.15 × 10 −5 s −1 ) 29 , and 9.4 nM (k a = 7.25 × 10 3 M −1 s −1 ; k d = 6.5 × 10 −5 s ...