title: Human epidermal growth factor receptor 2 (HER2) immunoreactivity: specificity of three pharmacodiagnostic antibodies.
abstract: OBJECTIVE
  The availability of specific antibody-based test systems is essential to testing of HER2 protein expression. Here, we mapped epitopes recognized by three pharmacodiagnostic HER2 antibodies and investigated their specificity towards peptides and fusion proteins homologous to the intracellular domains of HER1, HER2, HER3 and HER4. The investigated antibodies were PATHWAY(®) HER2 (clone 4B5; Ventana Medical Systems Inc., Tucson, AZ, USA), HercepTest™ (Dako Denmark A/S, Glostrup, Denmark), and Oracle(®) HER2 (clone CB11; Leica Microsystems GmbH, Wetzlar, Germany).
  
  
  RESULTS
  Epitopes were mapped using the alanine scanning method. Specificity was investigated in immunohistochemical stainings, competitive enzyme-linked immunosorbent assay (ELISA) and immunoblotting. All three antibodies reacted with HER2 proteins and peptides in immunohistochemical stainings, ELISA and immunoblotting. PATHWAY(®) HER2 also stained HER4-expressing cells, reacted with HER4 peptide in ELISA and detected HER4 fusion protein in an immunoblot. Oracle(®) HER2 weakly detected HER4 in immunohistochemical stainings, whereas the HercepTest™ antibody showed no cross-reactivity with other HER proteins.
  
  
  CONCLUSIONS
  Our study shows that the PATHWAY(®) HER2 antibody can bind HER4 peptides and fusion proteins in three different experimental settings. This should be investigated further to determine whether binding of HER4 also occurs in tissue samples and if such binding would have implications for therapy decisions for breast cancer patients.
authors.affiliation: The Faculty of Life Sciences, Institute for Veterinary Disease Biology and Sino-Danish Breast Cancer Research Centre, University of Copenhagen, Frederiksberg, Denmark. sofie@life.ku.dk
authors.forename: Anne-Sofie
authors.initials: AS
authors.lastname: Schrohl
authors.forename: Hans Christian
authors.initials: HC
authors.lastname: Pedersen
authors.forename: Sussie Steen
authors.initials: SS
authors.lastname: Jensen
authors.forename: Signe Lykke
authors.initials: SL
authors.lastname: Nielsen
authors.forename: Nils
authors.initials: N
authors.lastname: Brünner
mesh_terms: D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D001943:Breast Neoplasms / Q000378:metabolism*; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D066246:ErbB Receptors / Q000032:analysis / Q000737:chemistry / Q000276:immunology; D005260:Female; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D066247:Receptor, ErbB-4
chemical_list: D000911:Antibodies, Monoclonal; D066246:ErbB Receptors; D018719:Erb-b2 Receptor Tyrosine Kinases; D066247:Receptor, ErbB-4; C512478:EGFR protein, human; C508053:ERBB2 protein, human; C587689:ERBB4 protein, human
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