NDLI: Selection of recombinant anti-SH3 domain antibodies by high-throughput phage display

Content Provider	World Health Organization (WHO)-Global Index Medicus
Author	Jarvik, Nick Nadeem, Vincent Sidhu, Sachdev S. Moffat, Jason Economopoulos, Nicolas O. Huang, Haiming Uetrecht, Andrea Pawson, Tony Gu, Jun Liu, Bernard A. Miersch, Shane
Description	Author Affiliation: Huang H ( The Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario, Canada, M5S 3E1.); Economopoulos NO ( Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario, Canada, M5S 3E1.); Liu BA ( The Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario, Canada, M5S 3E1.); Uetrecht A ( Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario, Canada, M5S 3E1.); Gu J ( Samuel Lunenfeld Research Institute, Mount Sinai Hospital, University of Toronto, Toronto, Ontario, Canada, M5S 3E1.); Jarvik N ( Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada, M5S 3E1.); Nadeem V ( The Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario, Canada, M5S 3E1.); Pawson T ( Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada, M5S 3E1.); Moffat J ( The Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario, Canada, M5S 3E1.); Miersch S ( Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario, Canada, M5S 3E1.); Sidhu SS ( The Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario, Canada, M5S 3E1.)
Abstract	Antibodies are indispensable tools in biochemical research and play an expanding role as therapeutics. While hybridoma technology is the dominant method for antibody production, phage display is an emerging technology. Here, we developed and employed a high-throughput pipeline that enables selection of antibodies against hundreds of antigens in parallel. Binding selections using a phage-displayed synthetic antigen-binding fragment (Fab) library against 110 human SH3 domains yielded hundreds of Fabs targeting 58 antigens. Affinity assays demonstrated that representative Fabs bind tightly and specifically to their targets. Furthermore, we developed an efficient affinity maturation strategy adaptable to high-throughput, which increased affinity dramatically but did not compromise specificity. Finally, we tested Fabs in common cell biology applications and confirmed recognition of the full-length antigen in immunoprecipitation, immunoblotting and immunofluorescence assays. In summary, we have established a rapid and robust high-throughput methodology that can be applied to generate highly functional and renewable antibodies targeting protein domains on a proteome-wide scale.
ISSN	09618368
e-ISSN	1469896X
Journal	Protein Science
Issue Number	11
Volume Number	24
Language	English
Publisher	Wiley-Blackwell (on behalf of The Protein Society)
Publisher Date	2015-11-01
Publisher Place	United States
Access Restriction	Open
Subject Keyword	Antibodies Chemistry Cell Surface Display Techniques High-throughput Screening Assays Recombinant Fusion Proteins Src Homology Domains Genetics Amino Acid Sequence Metabolism Hek293 Cells Molecular Sequence Data Protein Engineering Sequence Alignment Research Support, Non-u.s. Gov't Discipline Biochemistry
Content Type	Text
Resource Type	Article
Subject	Medicine Molecular Biology Biochemistry

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