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).
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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.
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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.
Integrated supplementary information
Supplementary 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.
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 ± S.D.DAfp = 58.36 ± 79.18, nDFA=174; Mean ± S.DFFfp= 89.06 ± 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.
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.
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–5 and Supplementary Note 1
Supplementary Table 1
List of proteins recognized by the Xist probe on HuProt
Supplementary Table 2
List of approved targets for the PCRP project
Supplementary Table 3
List of recombinant domain antigens produced in E. coli
Supplementary Table 4
List of recombinant full-length proteins produced in yeast
Supplementary Table 5
List of commercially sourced mAbs and PCRP mAbs tested in a competitive IP protocol
Supplementary Table 6
Competitive IP analysis
Supplementary Table 7
List of mAbs that passed in ChIP-seq
Supplementary Table 8
List of all mAbs that passed HuProt, along with all recorded parameters
Supplementary Table 9
List of all targets in the approved target list with corresponding details on passing mAbs at different stages of the pipeline
Supplementary Table 10
List of all parameters and groups used in comparisons for the meta-analysis
Supplementary Table 11
Parametric comparisons and corresponding P values for mAbs generated by immunization with a single versus multiple antigens
Supplementary Table 12
Parametric comparisons and corresponding P values at the levels of targets
Supplementary Table 13
Parametric comparisons and corresponding P values for mAbs generated by intraperitoneal (i.p.) versus footpad (f.p.) immunization
Supplementary Table 14
Parametric comparisons and corresponding P values at the levels of targets
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
Supplementary Table 16
Parametric comparisons and corresponding P values at the levels of targets
Supplementary Table 17
List of IB+ and IB− mAbs that were tested and the corresponding parameters measured for these mAbs on denatured HuProt
Supplementary Table 18
Summary of mAbs at different stages of the pipeline by target class/subclass
Supplementary Table 19
Summary and success rates of targets at different stages of the pipeline classified by target class/subclass
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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Venkataraman, A., Yang, K., Irizarry, J. et al. A toolbox of immunoprecipitation-grade monoclonal antibodies to human transcription factors. Nat Methods 15, 330–338 (2018). https://doi.org/10.1038/nmeth.4632
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DOI: https://doi.org/10.1038/nmeth.4632
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