TY - JOUR
T1 - ELISA assay employing epitope-specific monoclonal antibodies to quantify circulating HER2 with potential application in monitoring cancer patients undergoing therapy with trastuzumab
AU - Agnolon, Valentina
AU - Contato, Anna
AU - Meneghello, Anna
AU - Tagliabue, Elda
AU - Toffoli, Giuseppe
AU - Gion, Massimo
AU - Polo, Federico
AU - Fabricio, Aline S.C.
N1 - Funding Information:
The authors thank Prof. F. Mancin (Department of Chemistry, University of Padua, Italy) and Prof. G. Pasut, (Department of Pharmaceutical and Pharmacological Sciences, University of Padua, Italy) for granting access to the Biacore facilities. The monoclonal antibodies MGR2 and MGR3 are available upon request at the Istituto Nazionale dei Tumori (INT), Fondazione IRCCS, Molecular targeting Unit (Milan, Italy). The developed in-house sandwich ELISA assay is available for eventual collaborative studies at the Regional Centre for Biomarkers, Department of Clinical Pathology and Transfusion Medicine (ULSS3 Serenissima, Regional Hospital, Venice, Italy). This work was supported by the Associazione Italiana per la Ricerca sul Cancro (AIRC) under the grant assigned for the Project 12214 (Innovative Tools for cancer risk assessment and early diagnosis − 5 × 1000) and the Regione Friuli-Venezia-Giulia under the grant assigned for the Project “Nano Diagnostic and Automated Therapeutic Tools for Oncology” (NADIATools – POR-FESR 2016, call 1.3b Smart Health).
Publisher Copyright:
© 2020, The Author(s).
Copyright:
Copyright 2020 Elsevier B.V., All rights reserved.
PY - 2020/12/1
Y1 - 2020/12/1
N2 - 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.
AB - 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.
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U2 - 10.1038/s41598-020-59630-y
DO - 10.1038/s41598-020-59630-y
M3 - Article
VL - 10
JO - Scientific Reports
JF - Scientific Reports
SN - 2045-2322
IS - 1
M1 - 3016
ER -