Anand Venkataraman 1,iD, Kun Yang 2, Jose Irizarry 3, Mark Mackiewicz 4,iD, Paolo Mita 5,6,7,iD, Zheng Kuang 6,19,iD, Lin Xue 1, Devlina Ghosh 1, Shuang Liu 8, Pedro Ramos 3, Shaohui Hu 3, Diane Bayron Kain 3,19, Sarah Keegan 5,6, Richard Saul 8, Simona Colantonio 9, Hongyan Zhang 8, Florencia Pauli-Behn 4, Guang Song 8, Edisa Albino 3, Lillyann Asencio 3, Leonardo Ramos 3, Luvir Lugo 3, Gloriner Morell 3, Javier Rivera 3, Kimberly Ruiz 3, Ruth Almodovar 3, Luis Nazario 3, Keven Murphy 3, Ivan Vargas 3, Zully Ann Rivera-Pacheco 3, Christian Rosa 3, Moises Vargas 3, Jessica McDade 7, Brian S Clark 1, Sooyeon Yoo 1, Seva G Khambadkone 10, Jimmy de Melo 1, Milanka Stevanovic 1, Lizhi Jiang 1, Yana Li 11, Wendy Y Yap 3, Brittany Jones 12, Atul Tandon 12, Elliot Campbell 13,14, Gaetano T Montelione 13,14, Stephen Anderson 13,14, Richard M Myers 4, Jef D Boeke 5,6,7, David Fenyö 5,6, Gordon Whiteley 9, Joel S Bader 2, Ignacio Pino 3,iD, Daniel J Eichinger 3,iD, Heng Zhu 8,15,iD, Seth Blackshaw
Posted: March 19, 2018
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 https://proteincapture.org.
NeoBiotechnologies’ products were used in this study:
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.
Publication History:
Published in final edited form as: Nat Methods. 2018 Mar 19;15(5):330–338. doi: 10.1038/nmeth.4632
Footnotes:
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.
