title: A roadmap to generate renewable protein binders to the human proteome.
abstract: 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.
authors.affiliation: Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada. colwill@lunenfeld.ca
authors.forename: Karen
authors.initials: K
authors.lastname: Colwill
authors.forename: Susanne
authors.initials: S
authors.lastname: Gräslund
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D002460:Cell Line; D004285:Dogs; D004797:Enzyme-Linked Immunosorbent Assay; D005455:Fluorescent Antibody Technique; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D015337:Multicenter Studies as Topic; D040081:Protein Array Analysis; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry* / Q000276:immunology*; D020543:Proteome / Q000737:chemistry / Q000276:immunology*; D011994:Recombinant Proteins / Q000737:chemistry; D020349:Surface Plasmon Resonance; D018909:src Homology Domains / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D011506:Proteins; D020543:Proteome; D011994:Recombinant Proteins