A High Through-put Platform for Recombinant Antibodies to Folded Proteins

Antibodies are key reagents in biology and medicine, but commercial sources are rarely recombinant and thus do not provide a permanent and renewable resource. Here, we describe an industrialized platform to generate antigens and validated recombinant antibodies for 346 transcription factors (TFs) and 211 epigenetic antigens. We describe an optimized automated phage display and antigen expression pipeline that in aggregate produced about 3000 sequenced Fragment antigen-binding domain that had high affinity (typically EC50<20 nm), high stability (Tm∼80 °C), good expression in E. coli (∼5 mg/L), and ability to bind antigen in complex cell lysates. We evaluated a subset of Fabs generated to homologous SCAN domains for binding specificities. These Fragment antigen-binding domains were monospecific to their target SCAN antigen except in rare cases where they cross-reacted with a few highly related antigens. Remarkably, immunofluorescence experiments in six cell lines for 270 of the TF antigens, each having multiple antibodies, show that ∼70% stain predominantly in the cytosol and ∼20% stain in the nucleus which reinforces the dominant role that translocation plays in TF biology. These cloned antibody reagents are being made available to the academic community through our web site recombinant-antibodies.org to allow a more system-wide analysis of TF and chromatin biology. We believe these platforms, infrastructure, and automated approaches will facilitate the next generation of renewable antibody reagents to the human proteome in the coming decade. Antibodies are key reagents in biology and medicine, but commercial sources are rarely recombinant and thus do not provide a permanent and renewable resource. Here, we describe an industrialized platform to generate antigens and validated recombinant antibodies for 346 transcription factors (TFs) and 211 epigenetic antigens. We describe an optimized automated phage display and antigen expression pipeline that in aggregate produced about 3000 sequenced Fragment antigen-binding domain that had high affinity (typically EC50<20 nm), high stability (Tm∼80 °C), good expression in E. coli (∼5 mg/L), and ability to bind antigen in complex cell lysates. We evaluated a subset of Fabs generated to homologous SCAN domains for binding specificities. These Fragment antigen-binding domains were monospecific to their target SCAN antigen except in rare cases where they cross-reacted with a few highly related antigens. Remarkably, immunofluorescence experiments in six cell lines for 270 of the TF antigens, each having multiple antibodies, show that ∼70% stain predominantly in the cytosol and ∼20% stain in the nucleus which reinforces the dominant role that translocation plays in TF biology. These cloned antibody reagents are being made available to the academic community through our web site recombinant-antibodies.org to allow a more system-wide analysis of TF and chromatin biology. We believe these platforms, infrastructure, and automated approaches will facilitate the next generation of renewable antibody reagents to the human proteome in the coming decade. Antibodies are crucial reagents for biological research and therapeutics. However, reproducibility for antibody reagents is a major concern, especially for polyclonals and even monoclonals where genetic drift of hybridoma stocks can be problematic (1.Pasqualini R. Arap W. Hybridoma-free generation of monoclonal antibodies.Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 257-259Crossref PubMed Scopus (49) Google Scholar, 2.Harlow E. Lane D. Using antibodies : a laboratory manual. 1999; xiv: 495Google Scholar). Moreover, some have estimated that less than half of the animal derived antibodies bind their cognate native proteins (3.Berglund L. Bjorling E. Oksvold P. Fagerberg L. Asplund A. Szigyarto C.A. Persson A. Ottosson J. Wernerus H. Nilsson P. Lundberg E. Sivertsson A. Navani S. Wester K. Kampf C. Hober S. Ponten F. Uhlen M. A genecentric Human Protein Atlas for expression profiles based PubMed Scopus Google Scholar, J. A. S. E. M. J. D. PubMed Scopus Google Scholar). generation of recombinant antibodies provide a renewable of cloned and highly validated antibody and a permanent A. A. antibodies in PubMed Scopus Google Scholar, S. D. Bjorling E. E. C.A. J. A. S. R. L. S. M. L. S. H. S. M. Uhlen M. S. C. P. H. A community for the of affinity PubMed Scopus Google Scholar). antibodies a for and to generate and for renewable antibody reagents J. S. A. S. M. W. A. J. J. of phage display to high antibody generation and PubMed Scopus Google Scholar, L. K. 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