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2018年3月19日NeoBiotechnologies, Inc., Union City, California, USA Brittany Jones & Atul Tandon Department of Molecular Biology and Biochemistry, Rutgers Universi...

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript. AbstractA key component of efforts to address the reproducibility crisis in biomedical research is the development of rigorously validated and renewable protein-affinity reagents. As part of the US National Institutes of Health (NIH) Protein Capture Reagents Program (PCRP), we have generated a collection of 1,406 highly validated immunoprecipitation- and/or immunoblotting-grade mouse monoclonal antibodies (mAbs) to 737 human transcription factors, using an integrated production and validation pipeline. We used HuProt human protein microarrays as a primary validation tool to identify mAbs with high specificity for their cognate targets. We further validated PCRP mAbs by means of multiple experimental applications, including immunoprecipitation, immunoblotting, chromatin immunoprecipitation followed by sequencing (ChIP-seq), and immunohistochemistry. We also conducted a meta-analysis that identified critical variables that contribute to the generation of high-quality mAbs. All validation data, protocols, and links to PCRP mAb suppliers are available at http://proteincapture.org. Subscription info for Chinese customersWe have a dedicated website for our Chinese customers. Please go to naturechina.com to subscribe to this journal.Go to naturechina.comRent or Buy articleGet time limited or full article access on ReadCube.from$8.99Rent or BuyAll prices are NET prices. References1Bradbury, A. Pl眉ckthun, A. Reproducibility: standardize antibodies used in research. Nature 518, 27鈥?9 (2015).CAS聽 Article聽 Google Scholar聽 2Weller, M.G. Quality issues of research antibodies. Anal. Chem. 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Landers lab (MIT, Cambridge, Massachusetts, USA).Author informationAuthor notesZheng KuangPresent address: Department of Immunology, University of Texas Southwestern Medical Center, Dallas, Texas, USADiane Bayron KainPresent address: BioReliance, Sigma-Aldrich Corp., Rockville, Maryland, USAAffiliationsSolomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland, USAAnand Venkataraman,聽Lin Xue,聽Devlina Ghosh,聽Brian S Clark,聽Sooyeon Yoo,聽Jimmy de Melo,聽Milanka Stevanovic,聽Lizhi Jiang聽 聽Seth BlackshawDepartment of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, USAKun Yang聽 聽Joel S BaderCDI Laboratories, Mayaguez, PR, USAJose Irizarry,聽Pedro Ramos,聽Shaohui Hu,聽Diane Bayron Kain,聽Edisa Albino,聽Lillyann Asencio,聽Leonardo Ramos,聽Luvir Lugo,聽Gloriner Morell,聽Javier Rivera,聽Kimberly Ruiz,聽Ruth Almodovar,聽Luis Nazario,聽Keven Murphy,聽Ivan Vargas,聽Zully Ann Rivera-Pacheco,聽Christian Rosa,聽Moises Vargas,聽Wendy Y Yap,聽Ignacio Pino聽 聽Daniel J EichingerHudsonAlpha Institute for Biotechnology, Huntsville, Alabama, USAMark Mackiewicz,聽Florencia Pauli Behn聽 聽Richard M MyersInstitute for System Genetics, NYU Langone Health, New York, New York, USAPaolo Mita,聽Sarah Keegan,聽Jef D Boeke聽 聽David Feny枚Department of Biochemistry and Molecular Pharmacology, NYU Langone Health, New York, New York, USAPaolo Mita,聽Zheng Kuang,聽Sarah Keegan,聽Jef D Boeke聽 聽David Feny枚Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland, USAPaolo Mita,聽Jessica McDade聽 聽Jef D BoekeDepartment of Pharmacology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USAShuang Liu,聽Richard Saul,聽Hongyan Zhang,聽Guang Song聽 聽Heng ZhuCancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, Maryland, USASimona Colantonio聽 聽Gordon WhiteleyDepartment of Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland, USASeva G KhambadkoneEukaryotic Tissue Culture Facility, Johns Hopkins University School of Medicine, Baltimore, Maryland, USAYana LiNeoBiotechnologies, Inc., Union City, California, USABrittany Jones聽 聽Atul TandonDepartment of Molecular Biology and Biochemistry, Rutgers University, Piscataway, New Jersey, USAElliot Campbell,聽Gaetano T Montelione聽 聽Stephen AndersonCenter for Advanced Biotechnology and Medicine, Piscataway, New Jersey, USAElliot Campbell,聽Gaetano T Montelione聽 聽Stephen AndersonCenter for Human Systems Biology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USAHeng Zhu聽 聽Seth BlackshawDepartment of Ophthalmology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USASeth BlackshawDepartment of Neurology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USASeth BlackshawInstitute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, USASeth BlackshawAuthorsAnand VenkataramanView author publicationsYou can also search for this author in PubMed聽Google ScholarKun YangView author publicationsYou can also search for this author in PubMed聽Google ScholarJose IrizarryView author publicationsYou can also search for this author in PubMed聽Google ScholarMark MackiewiczView author publicationsYou can also search for this author in PubMed聽Google ScholarPaolo MitaView author publicationsYou can also search for this author in PubMed聽Google ScholarZheng KuangView author publicationsYou can also search for this author in PubMed聽Google ScholarLin XueView author publicationsYou can also search for this author in PubMed聽Google ScholarDevlina GhoshView author publicationsYou can also search for this author in PubMed聽Google ScholarShuang LiuView author publicationsYou can also search for this author in PubMed聽Google ScholarPedro RamosView author publicationsYou can also search for this author in PubMed聽Google ScholarShaohui HuView author publicationsYou can also search for this author in PubMed聽Google ScholarDiane Bayron KainView author publicationsYou can also search for this author in PubMed聽Google ScholarSarah KeeganView author publicationsYou can also search for this author in PubMed聽Google ScholarRichard SaulView author 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BlackshawView author publicationsYou can also search for this author in PubMed聽Google ScholarContributionsA.V., M.M., P.M., Z.K., L.X., Y.L., D.G., S.L., P.R., S.H., D.B.K., H. Zhang, F.P.-B., G.S., E.A., L.A., L.R., L.L., G.M., J.R., K.R., R.A., L.N., K.M., I.V., Z.A.R.-P., C.R., M.V., J.M., B.S.C., S.Y., S.G.K., J.d.M., M.S., L.J., B.J., A.T. and E.C. performed experimental work. R.S. and S.C. performed independent validation of PCRP mAbs. K.Y., J.I. and S.K. designed algorithms and implemented software. W.Y.Y., S.A., G.T.M., R.M.M., J.D.B., D.F., G.W., D.J.E., J.S.B., I.P., H. Zhu and S.B. contributed expertise and supervision. All authors contributed to manuscript preparation.Corresponding authorsCorrespondence to Ignacio Pino or Daniel J Eichinger or Heng Zhu or Seth Blackshaw.Ethics declarations Competing interests S.B., H. Zhu, I.P., D.J.E., and J.D.B. are cofounders and shareholders of CDI Labs Inc. J.I., P.R., D.B.K., E.A., L.A., L.R., L.L., G.M., J.R., K.R., R.A., L.N., K.M., I.V., Z.A.R.-P., C.R., M.V., and W.Y.Y. are employees of CDI Labs Inc. A.V. and J.D.B. are consultants to CDI Labs Inc. J.D.B. serves on the Board of Directors of CDI Labs, and J.D.B.s relationship with CDI Labs is managed by NYU Langone Healths committee on conflicts of interest. B.J. is an employee of NeoBiotechnologies, Inc. A.T. is the founder and sole owner of NeoBiotechnologies, Inc. G.T.M. is founder and shareholder of Nexomics Biosciences, Inc. Integrated supplementary informationSupplementary Figure 1 HuProt proteins are in native conformation.HuProt proteins are in native conformation. (a) Categories of proteins based on GO annotation as represented on HuProt compared to the entire human proteome as per Uniprot. (b) Top panels. Anti-ACO2 6D1BE4 mAb (Catalog #ab110320, Abcam), which recognizes a conformational specific epitope, tested for target binding on a native HuProt (left) and HuProt denatured with 9M Urea and 5 mM DTT (right). Bottom panels. Anti-SMAD4 mAb (CDI Labs, #R516.2.1G11), which recognizes a linear epitope, tested for target binding on native HuProt (left) and denatured HuProt (right). Data shown here is representative of three independent technical replicates. (c,d) A conformation-specific anti-GST antibody (CDI Labs, #27.3.6G8) tested for binding to GST-tagged proteins on the native HuProt (c, top) and denatured HuProt (c, bottom).(d) Scatter plot of signal intensity measured with the conformation-specific anti-GST antibody (CDI Labs, #27.3.6G8) in native versus denatured HuProt (e) Micrographs representing the HuProt signal observed for known Xist interaction partner (HNRNPC) and potential new partners identified in our screen (RBM46 and ELAVL2). (f) Scatter plot of signal intensity measured with Xist probe on native HuProt versus a denatured HuProt. Source data Supplementary Figure 2 Competitive IP analysis.Commercially sourced antibodies when tested on HuProt exhibit interactions with targets other than the intended target. These antibodies also interact with the off-targets in a competitive immunoprecipitation experiment. Rank, z and S-score for off-targets represented here are indicated in Table S5. Antibodies tested are as follows: (a,b) Novus Biological anti-RELA (catalog# NB100-56055, lot# AB071609E) and PCRP anti-RELA (#YP268.1.2B6). (c) Santa Cruz Biotechnology anti-FOSL1 (Catalog# SC-28310, Lot# I1415) and PCRP anti-FOSL1 (clone ID# R1024.1.1G1). (d,e) Cell Signaling Technologies anti-FOSL1 (Catalog#5281, Lot#2) and PCRP anti-FOSL1 (#R1024.1.1G1). (f) Abgent anti-USF2 (Catalog# AT4478a, Lot# 11189) and PCRP anti-USF2 (clone ID# R1156.1.1A7). (g) LifeSpan Biotechnology anti-ZEB2 (Catalog# LS-C175748, Lot# 51937). * indicated in the immunoblots refer to unidentified off-targets detected by the commercial mAbs.Supplementary Figure 3 IHC staining of paraffin-embedded human tissue.IHC staining using clinical gold standard for diagnosing cancer in (a) colon (anti-P53, clone ID# BP53-12, NeoBiotechnologies), (b) pancreas (anti-SOX9, clone ID# 3B10.1F9, NeoBiotechnologies) and (c) colon (anti-CDX2, Clone ID #1690, NeoBiotechnologies). IHC staining using PCRP mAbs graded as true positive by certified clinical pathologist in cancerous tissue of (d) colon (anti-P53, clone ID# JH66.2.2A10), (e) pancreas (anti-SOX9, clone ID# YP73.1.1A2) and (f) colon (anti-CDX2, clone ID #R1435.1.1A3). IHC staining with anti-CDX2 (clone ID# R1435.1.1A3) shows no detectable signal in human cancer tissue from (g) liver,(h) skeletal-muscle, (i) prostate, (j) ovary, (k) skin and (l) lungs. IHC staining with anti-STAT3 (clone ID# R1231.1.2F12) allows detection of this nearly ubiquitously expressed target in human cancers of the (m) colon, (n) kidney, (o) lung, (p) ovary and (q) uterus. (r) IHC staining with anti-STAT3 (clone ID# R1231.1.2F12) exhibits no discernible signal in human skeletal muscle. Images are captured at 200x magnification.Supplementary Figure 4 Full-length (FL) immunogens provide efficiencies comparable to those of domain antigens.Key to the graphs represent abbreviations for mAb grouping using a single immunogen for immunization (see online methods for details). D-A-f.p. =Domain-All-footpad. F-F-f.p. = Full length-Full length- footpad (a) z-/S- score are higher in D-A-f.p. (n=174) versus the F-F-f.p (n=530) mAbs. Mean 卤 S.D.DAfp = 58.36 卤 79.18, nDFA=174; Mean 卤 S.DFFfp= 89.06 卤 32.05, nDDA=530. * and *** represents p=0.01 and 1.39E-06 respectively by Benjamini-Hochberg FDR (b) Comparing success rates of D-A-f.p. (n=33) and F-F-f.p (n=215) mAbs at different stages of the validation pipeline. Source data Supplementary Figure 5 Summary of HuProt+ mAbs.HuProt+ mAbs that were tested and passed IP and/or IB by (a) mAbs (b) targets classified into target class. Source data Supplementary information Supplementary Text and FiguresSupplementary Figures 1鈥? and Supplementary Note 1 Life Sciences Reporting Summary Supplementary Table 1List of proteins recognized by the Xist probe on HuProt Supplementary Table 2List of approved targets for the PCRP project Supplementary Table 3List of recombinant domain antigens produced in E. coli Supplementary Table 4List of recombinant full-length proteins produced in yeast Supplementary Table 5List of commercially sourced mAbs and PCRP mAbs tested in a competitive IP protocol Supplementary Table 6Competitive IP analysis Supplementary Table 7List of mAbs that passed in ChIP-seq Supplementary Table 8List of all mAbs that passed HuProt, along with all recorded parameters Supplementary Table 9List of all targets in the approved target list with corresponding details on passing mAbs at different stages of the pipeline Supplementary Table 10List of all parameters and groups used in comparisons for the meta-analysis Supplementary Table 11Parametric comparisons and corresponding P values for mAbs generated by immunization with a single versus multiple antigens Supplementary Table 12Parametric comparisons and corresponding P values at the levels of targets Supplementary Table 13Parametric comparisons and corresponding P values for mAbs generated by intraperitoneal (i.p.) versus footpad (f.p.) immunization Supplementary Table 14Parametric comparisons and corresponding P values at the levels of targets Supplementary Table 15Parametric comparisons and corresponding P values between mAbs that recognize only their cognate immunized domain and those that recognize their intended full-length target on HuProt Supplementary Table 16Parametric comparisons and corresponding P values at the levels of targets Supplementary Table 17List of IB+ and IB鈭?mAbs that were tested and the corresponding parameters measured for these mAbs on denatured HuProt Supplementary Table 18Summary of mAbs at different stages of the pipeline by target class/subclass Supplementary Table 19Summary and success rates of targets at different stages of the pipeline classified by target class/subclass Supplementary Table 20Summary of targets and success rates at different stages of the pipeline classified by type and route of immunization used to generate the mAbsSource data He-wei Jiang, Yang Li, Hai-nan Zhang, Wei Wang, Xiao Yang, Huan Qi, Hua Li, Dong Men, Jie Zhou Sheng-ce Tao Nature Communications (2020) Isha S. Dhande, Sterling C. Kneedler, Yaming Zhu, Aniket S. Joshi, M. John Hicks, Scott E. Wenderfer, Michael C. Braun Peter A. Doris Genes Immunity (2020) Claudia Fredolini, Sanna Bystr枚m, Laura Sanchez-Rivera, Marina Ioannou, Davide Tamburro, Fredrik Pont茅n, Rui M. Branca, Peter Nilsson, Janne Lehti枚 Jochen M. Schwenk Scientific Reports (2019) Sign up for the Nature Briefing newsletter 鈥?what matters in science, free to your inbox daily.