Despite the wealth of commercially available antibodies to human proteins, research is often hindered by their inconsistent validation, their poor performance and the inadequate coverage of the proteome. These issues could be addressed by systematic, genome-wide efforts to generate and validate renewable protein binders. We report a multicenter study to assess the potential of hybridoma and phage-display technologies in a coordinated large-scale antibody generation and validation effort. We produced over 1,000 antibodies targeting 20 SH2 domain proteins and evaluated them for potency and specificity by enzyme-linked immunosorbent assay (ELISA), protein microarray and surface plasmon resonance (SPR). We also tested selected antibodies in immunoprecipitation, immunoblotting and immunofluorescence assays. Our results show that high-affinity, high-specificity renewable antibodies generated by different technologies can be produced quickly and efficiently. We believe that this work serves as a foundation and template for future larger-scale studies to create renewable protein binders.
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We acknowledge B. Liu and P. Nash for providing SH2 domain alignments; N. Bisson (Samuel Lunenfeld Research Institute) for providing a stable cell line encoding Flag-tagged Grb2; M. Taussig and M. Sundstrom for helpful discussions; and N. Bisson and B. Liu for critically reading the manuscript. The Structural Genomics Consortium is a registered charity (number 1097737) that receives funds from the Canadian Institutes for Health Research, the Canadian Foundation for Innovation, Genome Canada through the Ontario Genomics Institute, GlaxoSmithKline, Karolinska Institutet, the Knut and Alice Wallenberg Foundation, the Ontario Innovation Trust, the Ontario Ministry for Research and Innovation, Merck & Co., Inc., the Novartis Research Foundation, the Swedish Agency for Innovation Systems, the Swedish Foundation for Strategic Research and the Wellcome Trust. This work was also supported by funds from Genome Canada through the Ontario Genomics Institute and Ontario Research Fund Global Leadership Round in Genomics and Life Sciences (to T.P.), The Human Frontier Science Program (O.R.), the European Commission 6th framework program coordination action 'Proteome Binders' (S.D.), from the Systematisch-Methodischen Platform Antibody Factory, within the German Nationales Genomforschungsnetz (S.D.), the Land Niedersachsen (S.D.), the Wellcome Trust (J.Mc.), the US National Institutes of Health (GM082288-09A1 and EY016094-01A1 to B.K.K. and R01-GM72688 and U54-GM74946 to A.A.K. and S.K.), to the Swedish Research Council (524-2008-617) (H.P.), and the Victorian State Government (The Department of Innovation, Industry and Regional Development), the Australian Federal Government (Bioplatforms Australia) and Monash University for financial support for the Monash Antibody Technologies Facility.
The authors declare no competing financial interests.
A full list of authors appears at the end of this paper.
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