Selection of recombinant anti-SH3 domain antibodies by high-throughput phage display.
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.
The Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario, Canada, M5S 3E1.|Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario, Canada, M5S 3E1.
Haiming
H
Huang
The Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario, Canada, M5S 3E1.|Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario, Canada, M5S 3E1.
Nicolas O
NO
Economopoulos
Samuel Lunenfeld Research Institute, Mount Sinai Hospital, University of Toronto, Toronto, Ontario, Canada, M5S 3E1.|Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada, M5S 3E1.
Bernard A
BA
Liu
The Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario, Canada, M5S 3E1.|Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada, M5S 3E1.
Andrea
A
Uetrecht
The Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario, Canada, M5S 3E1.|Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario, Canada, M5S 3E1.
Jun
J
Gu
The Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario, Canada, M5S 3E1.
Nick
N
Jarvik
The Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario, Canada, M5S 3E1.|Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario, Canada, M5S 3E1.
Vincent
V
Nadeem
Samuel Lunenfeld Research Institute, Mount Sinai Hospital, University of Toronto, Toronto, Ontario, Canada, M5S 3E1.|Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada, M5S 3E1.
Tony
T
Pawson
The Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario, Canada, M5S 3E1.|Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada, M5S 3E1.
Jason
J
Moffat
The Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario, Canada, M5S 3E1.|Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario, Canada, M5S 3E1.
Shane
http://orcid.org/0000-0003-4785-4508
S
Miersch
The Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario, Canada, M5S 3E1.|Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario, Canada, M5S 3E1.|Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada, M5S 3E1.
Sachdev S
SS
Sidhu
D000595:Amino Acid Sequence; D000906:Antibodies / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D061505:Cell Surface Display Techniques / Q000379:methods*; D057809:HEK293 Cells; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D008969:Molecular Sequence Data; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D018909:src Homology Domains / Q000235:genetics*
D000906:Antibodies; D011993:Recombinant Fusion Proteins
SH3 domain; antibodies; high throughput method; phage display