Chemistry & biology, 2014-03-20, Vol.21 (3), p.357-368
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.
•Highly specific detection of protein-protein interactions in situ•Highly specific detection of protein phosphorylation in situ•Superior target specificity of a “dual binder” compared to monoclonal antibody
Protein-protein interactions and protein phosphorylation are crucial events in cell activation. In the “dual binder” technology developed by van Dieck et al., bispecific reagents are used to detect such events with high specificity. Dual binders achieve specificity through a cooperative binding effect.
Animals ; Antibodies - chemistry ; Antibodies - immunology ; Antigenic determinants ; Cell Line, Tumor ; Dimerization ; Formaldehyde ; HEK293 Cells ; Humans ; Immunoglobulin Fab Fragments - chemistry ; Immunoglobulin Fab Fragments - genetics ; Immunoglobulin Fab Fragments - metabolism ; Immunohistochemistry ; MCF-7 Cells ; Mice ; Monoclonal antibodies ; Oligonucleotides - chemical synthesis ; Oligonucleotides - chemistry ; Phosphorylation ; Protein Interaction Domains and Motifs ; Proteins ; Proteins - metabolism ; Receptor, ErbB-2 - analysis ; Receptor, ErbB-2 - genetics ; Receptor, ErbB-2 - metabolism ; Receptor, ErbB-3 - analysis ; Receptor, ErbB-3 - genetics ; Receptor, ErbB-3 - metabolism ; Recombinant Proteins - biosynthesis ; Recombinant Proteins - chemistry ; Recombinant Proteins - genetics
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