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Development of bispecific molecules for the in situ detection of protein-protein interactions and protein phosphorylation.
van Dieck, Jan; Schmid, Volker; Heindl, Dieter; Dziadek, Sebastian; Schraeml, Michael; Gerg, Michael; Massoner, Petra; Engel, Alfred M; Tiefenthaler, Georg; Vural, Serhat; Stritt, Simon; Tetzlaff, Fabian; Soukupova, Monika; Kopetzki, Erhard; Bossenmaier, Birgit; Thomas, Marlene; Klein, Christian; Mertens, Alfred; Heller, Astrid; Tacke, Michael.
Afiliação
  • van Dieck J; Pharma Research and Early Development, Roche Diagnostics GmbH, 82377 Penzberg, Germany.
  • Schmid V; Pharma Research and Early Development, Roche Diagnostics GmbH, 82377 Penzberg, Germany.
  • Heindl D; Roche Professional Diagnostics, Roche Diagnostics GmbH, 82377 Penzberg, Germany.
  • Dziadek S; Roche Professional Diagnostics, Roche Diagnostics GmbH, 82377 Penzberg, Germany.
  • Schraeml M; Roche Professional Diagnostics, Roche Diagnostics GmbH, 82377 Penzberg, Germany.
  • Gerg M; Roche Professional Diagnostics, Roche Diagnostics GmbH, 82377 Penzberg, Germany.
  • Massoner P; Roche Professional Diagnostics, Roche Diagnostics GmbH, 82377 Penzberg, Germany.
  • Engel AM; Roche Professional Diagnostics, Roche Diagnostics GmbH, 82377 Penzberg, Germany.
  • Tiefenthaler G; Pharma Research and Early Development, Roche Diagnostics GmbH, 82377 Penzberg, Germany.
  • Vural S; Roche Professional Diagnostics, Roche Diagnostics GmbH, 82377 Penzberg, Germany.
  • Stritt S; Pharma Research and Early Development, Roche Diagnostics GmbH, 82377 Penzberg, Germany.
  • Tetzlaff F; Pharma Research and Early Development, Roche Diagnostics GmbH, 82377 Penzberg, Germany.
  • Soukupova M; Roche Professional Diagnostics, Roche Diagnostics GmbH, 82377 Penzberg, Germany.
  • Kopetzki E; Pharma Research and Early Development, Roche Diagnostics GmbH, 82377 Penzberg, Germany.
  • Bossenmaier B; Pharma Research and Early Development, Roche Diagnostics GmbH, 82377 Penzberg, Germany.
  • Thomas M; Pharma Research and Early Development, Roche Diagnostics GmbH, 82377 Penzberg, Germany.
  • Klein C; Pharma Research and Early Development, Roche Glycart AG, 8952 Schlieren, Switzerland.
  • Mertens A; Pharma Research and Early Development, Roche Diagnostics GmbH, 82377 Penzberg, Germany.
  • Heller A; Pharma Research and Early Development, Roche Diagnostics GmbH, 82377 Penzberg, Germany.
  • Tacke M; Roche Professional Diagnostics, Roche Diagnostics GmbH, 82377 Penzberg, Germany. Electronic address: michael.tacke@roche.com.
Chem Biol ; 21(3): 357-68, 2014 Mar 20.
Article em En | MEDLINE | ID: mdl-24529991
Investigation of protein-protein interactions (PPIs) and protein phosphorylation in clinical tissue samples can offer valuable information about the activation status and function of proteins involved in disease progression. However, existing antibody-based methods for phosphorylation detection have been found to lack specificity, and methods developed for examining PPIs in vitro cannot be easily adapted for tissues samples. In this study, we eliminated some of these limitations by developing a specific immunohistochemical staining method that uses "dual binders" (DBs), which are bispecific detection agents consisting of two Fab fragment molecules joined by a flexible linker, to detect PPIs and protein phosphorylation. We engineered DBs by selecting Fab fragments with fast off-rate kinetics, which allowed us to demonstrate that stable target binding was achieved only upon simultaneous, cooperative binding to both epitopes. We show that DBs specifically detect the activated HER2/HER3 complex in formalin-fixed, paraffin-embedded cancer cells and exhibit superior detection specificity for phospho-HER3 compared to the corresponding monoclonal antibody. Overall, the performance of DBs makes them attractive tools for future development for clinical applications.
Assuntos

Texto completo: 1 Base de dados: MEDLINE Assunto principal: Imuno-Histoquímica / Proteínas / Receptor ErbB-2 / Receptor ErbB-3 Tipo de estudo: Diagnostic_studies Limite: Animals / Humans Idioma: En Ano de publicação: 2014 Tipo de documento: Article

Texto completo: 1 Base de dados: MEDLINE Assunto principal: Imuno-Histoquímica / Proteínas / Receptor ErbB-2 / Receptor ErbB-3 Tipo de estudo: Diagnostic_studies Limite: Animals / Humans Idioma: En Ano de publicação: 2014 Tipo de documento: Article