title: Antibody validation by combining immunohistochemistry and protein extraction from formalin-fixed paraffin-embedded tissues.
abstract: OBJECTIVE
  Personalized cancer treatment strategies depend on comprehensive and detailed characterization of individual human malignancies. Clinical pathology, particularly immunohistochemical evaluation of biomarkers in tissues, is considered to be the approved standard for diagnostic and therapeutic decisions, having a direct influence on patient management and therapy. Although antibody-based approaches are established and integrated successfully into both clinical and research applications, for personalized treatment regimens new demands have been placed on the quality, reproducibility and accuracy of antibody-based assays. To ensure the accuracy of specific antigen detection in immunohistochemistry, we introduce a novel approach for antibody validation.
  
  
  RESULTS
  In a tandem approach we used the same archival tissue of interest for antibody validation by combining extraction of immunoreactive proteins from formalin-fixed, paraffin-embedded tissue with Western blot analysis and immunohistochemistry. This procedure allows for specification of the antigen detected and for localization of the protein in the tissue. Of the 32 antibodies tested used in research and routine diagnostics, 19 showed reliable specificity in both assays.
  
  
  CONCLUSIONS
  This study emphasizes the advantage of combining suitable methods to ensure reproducibility and specific antigen detection. Based on our results, we propose a novel step-by-step strategy to validate antibody specificity and reduce variability of immunohistochemical results.
authors.affiliation: Institute of Pathology, Ludwig-Maximilians-Universität München, Munich, Germany.
authors.forename: Claudia
authors.initials: C
authors.lastname: Schuster
authors.forename: Katharina
authors.initials: K
authors.lastname: Malinowsky
authors.forename: Sibylle
authors.initials: S
authors.lastname: Liebmann
authors.forename: Daniela
authors.initials: D
authors.lastname: Berg
authors.forename: Claudia
authors.initials: C
authors.lastname: Wolff
authors.forename: Kai
authors.initials: K
authors.lastname: Tran
authors.forename: Christina
authors.initials: C
authors.lastname: Schott
authors.forename: Simone
authors.initials: S
authors.lastname: Reu
authors.forename: Jens
authors.initials: J
authors.lastname: Neumann
authors.forename: Claudius
authors.initials: C
authors.lastname: Faber
authors.forename: Heinz
authors.initials: H
authors.lastname: Höfler
authors.forename: Thomas
authors.initials: T
authors.lastname: Kirchner
authors.forename: Karl-Friedrich
authors.initials: KF
authors.lastname: Becker
authors.forename: Falk
authors.initials: F
authors.lastname: Hlubek
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry*; D000918:Antibody Specificity; D005404:Fixatives; D005557:Formaldehyde; D007150:Immunohistochemistry; D016612:Paraffin Embedding; D040081:Protein Array Analysis; D015203:Reproducibility of Results; D016707:Tissue Fixation / Q000379:methods
chemical_list: D000911:Antibodies, Monoclonal; D005404:Fixatives; D005557:Formaldehyde