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Protein Sci. 2015 Nov;24(11):1890-900. doi: 10.1002/pro.2799. Epub 2015 Sep 16.

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

Author information

1
The Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario, Canada, M5S 3E1.
2
Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario, Canada, M5S 3E1.
3
Samuel Lunenfeld Research Institute, Mount Sinai Hospital, University of Toronto, Toronto, Ontario, Canada, M5S 3E1.
4
Department of Molecular Genetics, 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.

KEYWORDS:

SH3 domain; antibodies; high throughput method; phage display

PMID:
26332758
PMCID:
PMC4622221
DOI:
10.1002/pro.2799
[Indexed for MEDLINE]
Free PMC Article

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