Abstract

A 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.

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Acknowledgements

This work was supported by the NIH Common Fund (awards U54HG006434 (to J.D.B., S.B., and H.Z.) and U01DC011485 (to S.A. and G.T.M.)). Cy5-UTP-incorporated cRNA probes of Xist produced by T7-directed transcription were a kind gift from E. Lander's lab (MIT, Cambridge, Massachusetts, USA).

Author information

Author notes

    • Zheng Kuang
    •  & Diane Bayron Kain

    Present addresses: Department of Immunology, University of Texas Southwestern Medical Center, Dallas, Texas, USA (Z.K.); BioReliance, Sigma-Aldrich Corp., Rockville, Maryland, USA (D.B.K.).

Affiliations

  1. Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

    • Anand Venkataraman
    • , Lin Xue
    • , Devlina Ghosh
    • , Brian S Clark
    • , Sooyeon Yoo
    • , Jimmy de Melo
    • , Milanka Stevanovic
    • , Lizhi Jiang
    •  & Seth Blackshaw
  2. Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

    • Kun Yang
    •  & Joel S Bader
  3. CDI Laboratories, Mayaguez, PR, USA.

    • Jose Irizarry
    • , Pedro Ramos
    • , Shaohui Hu
    • , Diane Bayron Kain
    • , Edisa Albino
    • , Lillyann Asencio
    • , Leonardo Ramos
    • , Luvir Lugo
    • , Gloriner Morell
    • , Javier Rivera
    • , Kimberly Ruiz
    • , Ruth Almodovar
    • , Luis Nazario
    • , Keven Murphy
    • , Ivan Vargas
    • , Zully Ann Rivera-Pacheco
    • , Christian Rosa
    • , Moises Vargas
    • , Wendy Y Yap
    • , Ignacio Pino
    •  & Daniel J Eichinger
  4. HudsonAlpha Institute for Biotechnology, Huntsville, Alabama, USA.

    • Mark Mackiewicz
    • , Florencia Pauli Behn
    •  & Richard M Myers
  5. Institute for System Genetics, NYU Langone Health, New York, New York, USA.

    • Paolo Mita
    • , Sarah Keegan
    • , Jef D Boeke
    •  & David Fenyö
  6. Department of Biochemistry and Molecular Pharmacology, NYU Langone Health, New York, New York, USA.

    • Paolo Mita
    • , Zheng Kuang
    • , Sarah Keegan
    • , Jef D Boeke
    •  & David Fenyö
  7. Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

    • Paolo Mita
    • , Jessica McDade
    •  & Jef D Boeke
  8. Department of Pharmacology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

    • Shuang Liu
    • , Richard Saul
    • , Hongyan Zhang
    • , Guang Song
    •  & Heng Zhu
  9. Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, Maryland, USA.

    • Simona Colantonio
    •  & Gordon Whiteley
  10. Department of Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

    • Seva G Khambadkone
  11. Eukaryotic Tissue Culture Facility, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

    • Yana Li
  12. NeoBiotechnologies, Inc., Union City, California, USA.

    • Brittany Jones
    •  & Atul Tandon
  13. Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, New Jersey, USA.

    • Elliot Campbell
    • , Gaetano T Montelione
    •  & Stephen Anderson
  14. Center for Advanced Biotechnology and Medicine, Piscataway, New Jersey, USA.

    • Elliot Campbell
    • , Gaetano T Montelione
    •  & Stephen Anderson
  15. Center for Human Systems Biology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

    • Heng Zhu
    •  & Seth Blackshaw
  16. Department of Ophthalmology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

    • Seth Blackshaw
  17. Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

    • Seth Blackshaw
  18. Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

    • Seth Blackshaw

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Contributions

A.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.

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 Health's 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.

Corresponding authors

Correspondence to Ignacio Pino or Daniel J Eichinger or Heng Zhu or Seth Blackshaw.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–5 and Supplementary Note 1

  2. 2.

    Life Sciences Reporting Summary

Excel files

  1. 1.

    Supplementary Table 1

    List of proteins recognized by the Xist probe on HuProt

  2. 2.

    Supplementary Table 2

    List of approved targets for the PCRP project

  3. 3.

    Supplementary Table 3

    List of recombinant domain antigens produced in E. coli

  4. 4.

    Supplementary Table 4

    List of recombinant full-length proteins produced in yeast

  5. 5.

    Supplementary Table 5

    List of commercially sourced mAbs and PCRP mAbs tested in a competitive IP protocol

  6. 6.

    Supplementary Table 6

    Competitive IP analysis

  7. 7.

    Supplementary Table 7

    List of mAbs that passed in ChIP-seq

  8. 8.

    Supplementary Table 8

    List of all mAbs that passed HuProt, along with all recorded parameters

  9. 9.

    Supplementary Table 9

    List of all targets in the approved target list with corresponding details on passing mAbs at different stages of the pipeline

  10. 10.

    Supplementary Table 10

    List of all parameters and groups used in comparisons for the meta-analysis

  11. 11.

    Supplementary Table 11

    Parametric comparisons and corresponding P values for mAbs generated by immunization with a single versus multiple antigens

  12. 12.

    Supplementary Table 12

    Parametric comparisons and corresponding P values at the levels of targets

  13. 13.

    Supplementary Table 13

    Parametric comparisons and corresponding P values for mAbs generated by intraperitoneal (i.p.) versus footpad (f.p.) immunization

  14. 14.

    Supplementary Table 14

    Parametric comparisons and corresponding P values at the levels of targets

  15. 15.

    Supplementary Table 15

    Parametric 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

  16. 16.

    Supplementary Table 16

    Parametric comparisons and corresponding P values at the levels of targets

  17. 17.

    Supplementary Table 17

    List of IB+ and IB− mAbs that were tested and the corresponding parameters measured for these mAbs on denatured HuProt

  18. 18.

    Supplementary Table 18

    Summary of mAbs at different stages of the pipeline by target class/subclass

  19. 19.

    Supplementary Table 19

    Summary and success rates of targets at different stages of the pipeline classified by target class/subclass

  20. 20.

    Supplementary Table 20

    Summary of targets and success rates at different stages of the pipeline classified by type and route of immunization used to generate the mAbs

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DOI

https://doi.org/10.1038/nmeth.4632