Skip to main content

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.

  • Article
  • Published:

A proteomics approach for the identification and cloning of monoclonal antibodies from serum

This article has been updated

Abstract

We describe a proteomics approach that identifies antigen-specific antibody sequences directly from circulating polyclonal antibodies in the serum of an immunized animal. The approach involves affinity purification of antibodies with high specific activity and then analyzing digested antibody fractions by nano-flow liquid chromatography coupled to tandem mass spectrometry. High-confidence peptide spectral matches of antibody variable regions are obtained by searching a reference database created by next-generation DNA sequencing of the B-cell immunoglobulin repertoire of the immunized animal. Finally, heavy and light chain sequences are paired and expressed as recombinant monoclonal antibodies. Using this technology, we isolated monoclonal antibodies for five antigens from the sera of immunized rabbits and mice. The antigen-specific activities of the monoclonal antibodies recapitulate or surpass those of the original affinity-purified polyclonal antibodies. This technology may aid the discovery and development of vaccines and antibody therapeutics, and help us gain a deeper understanding of the humoral response.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Overview of proteomics approach for identifying functionally relevant monoclonal antibodies from an immunized animal.
Figure 2: Affinity purification of progesterone receptor–specific polyclonal rabbit IgG.
Figure 3: Identification and characterization of functional monoclonal antibodies against progesterone receptor A/B.

Similar content being viewed by others

Change history

  • 28 March 2012

    In the version of this supplementary file originally posted online, the Figures and Tables were corrupted. The error has been corrected in this file as of 28 March 2012.

References

  1. Baltimore, D. Gene conversion: some implications for immunoglobulin genes. Cell 24, 592–594 (1981).

    Article  CAS  Google Scholar 

  2. Becker, R.S. & Knight, K.L. Somatic diversification of immunoglobulin heavy chain VDJ genes: evidence for somatic gene conversion in rabbits. Cell 63, 987–997 (1990).

    Article  CAS  Google Scholar 

  3. Hozumi, N. & Tonegawa, S. Evidence for somatic rearrangement of immunoglobulin genes coding for variable and constant regions. Proc. Natl. Acad. Sci. USA 73, 3628–3632 (1976).

    Article  CAS  Google Scholar 

  4. Kim, S., Davis, M., Sinn, E., Patten, P. & Hood, L. Antibody diversity: somatic hypermutation of rearranged VH genes. Cell 27, 573–581 (1981).

    Article  CAS  Google Scholar 

  5. Keller, M.A. & Stiehm, E.R. Passive immunity in prevention and treatment of infectious diseases. Clin. Microbiol. Rev. 13, 602–614 (2000).

    Article  CAS  Google Scholar 

  6. Lambert, J.S. & Stiehm, E.R. Passive immunity in the prevention of maternal-fetal transmission of human immunodeficiency virus infection. Ann. NY Acad. Sci. 693, 186–193 (1993).

    Article  CAS  Google Scholar 

  7. Köhler, G. & Milstein, C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256, 495–497 (1975).

    Article  Google Scholar 

  8. Barbas, C.F. III., Burton, D.R., Scott, J.K. & Silverman, G.J. Phage Display: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2001).

  9. Harlow, E. & Lane, D. Using Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1998).

  10. Lanzavecchia, A., Corti, D. & Sallusto, F. Human monoclonal antibodies by immortalization of memory B cells. Curr. Opin. Biotechnol. 18, 523–528 (2007).

    Article  CAS  Google Scholar 

  11. Meijer, P.J. et al. Isolation of human antibody repertoires with preservation of the natural heavy and light chain pairing. J. Mol. Biol. 358, 764–772 (2006).

    Article  CAS  Google Scholar 

  12. O'Brien, P.M. & Aitken, R. Antibody Phage Display: Methods and Protocols (Humana Press, 2002).

  13. Pasqualini, R. & Arap, W. Hybridoma-free generation of monoclonal antibodies. Proc. Natl. Acad. Sci. USA 101, 257–259 (2004).

    Article  CAS  Google Scholar 

  14. Sullivan, M., Kaur, K., Pauli, N. & Wilson, P.C. Harnessing the immune system's arsenal: producing human monoclonal antibodies for therapeutics and investigating immune responses. F1000 Biol. Rep. 3, 17 <http://f1000.com/reports/b/3/17> (2011).

    Article  Google Scholar 

  15. Walker, L.M. et al. Broad and potent neutralizing antibodies from an African donor reveal a new HIV-1 vaccine target. Science 326, 285–289 (2009).

    Article  CAS  Google Scholar 

  16. Fischer, N. Sequencing antibody repertoires: the next generation. MAbs 3, 17–20 (2011).

    Article  Google Scholar 

  17. Lakhani, S.R. et al. The pathology of familial breast cancer: predictive value of immunohistochemical markers estrogen receptor, progesterone receptor, HER-2, and p53 in patients with mutations in BRCA1 and BRCA2. J. Clin. Oncol. 20, 2310–2318 (2002).

    Article  CAS  Google Scholar 

  18. Becker, R.S., Suter, M. & Knight, K.L. Restricted utilization of VH and DH genes in leukemic rabbit B cells. Eur. J. Immunol. 20, 397–402 (1990).

    Article  CAS  Google Scholar 

  19. Knight, K.L. Restricted VH gene usage and generation of antibody diversity in rabbit. Annu. Rev. Immunol. 10, 593–616 (1992).

    Article  CAS  Google Scholar 

  20. Mage, R.G., Lanning, D. & Knight, K.L. B cell and antibody repertoire development in rabbits: the requirement of gut-associated lymphoid tissues. Dev. Comp. Immunol. 30, 137–153 (2006).

    Article  CAS  Google Scholar 

  21. Yates, J.R. III, Eng, J.K., McCormack, A.L. & Schieltz, D. Method to correlate tandem mass spectra of modified peptides to amino acid sequences in the protein database. Anal. Chem. 67, 1426–1436 (1995).

    Article  CAS  Google Scholar 

  22. Elias, J.E. & Gygi, S.P. Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry. Nat. Methods 4, 207–214 (2007).

    Article  CAS  Google Scholar 

  23. Huttlin, E.L. et al. A tissue-specific atlas of mouse protein phosphorylation and expression. Cell 143, 1174–1189 (2010).

    Article  CAS  Google Scholar 

  24. Kircher, M. & Kelso, J. High-throughput DNA sequencing–concepts and limitations. Bioessays 32, 524–536 (2010).

    Article  CAS  Google Scholar 

  25. Dereeper, A. et al. Phylogeny.fr: robust phylogenetic analysis for the non-specialist. Nucleic Acids Res. 36, W465–W469 (2008).

    Article  CAS  Google Scholar 

  26. Reddy, S.T. et al. Monoclonal antibodies isolated without screening by analyzing the variable-gene repertoire of plasma cells. Nat. Biotechnol. 28, 965–969 (2010).

    Article  CAS  Google Scholar 

  27. Bandeira, N., Pham, V., Pevzner, P., Arnott, D. & Lill, J.R. Automated de novo protein sequencing of monoclonal antibodies. Nat. Biotechnol. 26, 1336–1338 (2008).

    Article  CAS  Google Scholar 

  28. Castellana, N.E. et al. Resurrection of a clinical antibody: template proteogenomic de novo proteomic sequencing and reverse engineering of an anti-lymphotoxin-alpha antibody. Proteomics 11, 395–405 (2011).

    Article  CAS  Google Scholar 

  29. Rappsilber, J., Ishihama, Y. & Mann, M. Stop and go extraction tips for matrix-assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics. Anal. Chem. 75, 663–670 (2003).

    Article  CAS  Google Scholar 

  30. Villén, J. & Gygi, S.P. The SCX/IMAC enrichment approach for global phosphorylation analysis by mass spectrometry. Nat. Protoc. 3, 1630–1638 (2008).

    Article  Google Scholar 

  31. Beausoleil, S.A., Villen, J., Gerber, S.A., Rush, J. & Gygi, S.P. A probability-based approach for high-throughput protein phosphorylation analysis and site localization. Nat. Biotechnol. 24, 1285–1292 (2006).

    Article  CAS  Google Scholar 

  32. Wu, T.T. & Kabat, E.A. An analysis of the sequences of the variable regions of Bence Jones proteins and myeloma light chains and their implications for antibody complementarity. J. Exp. Med. 132, 211–250 (1970).

    Article  CAS  Google Scholar 

  33. Doronina, V.A. et al. Site-specific release of nascent chains from ribosomes at a sense codon. Mol. Cell. Biol. 28, 4227–4239 (2008).

    Article  CAS  Google Scholar 

  34. Donnelly, M.L. et al. The 'cleavage' activities of foot-and-mouth disease virus 2A site-directed mutants and naturally occurring '2A-like' sequences. J. Gen. Virol. 82, 1027–1041 (2001).

    Article  CAS  Google Scholar 

  35. Boussif, O. et al. A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proc. Natl. Acad. Sci. USA 92, 7297–7301 (1995).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We would like to dedicate this work to the memories of César Milstein and George Kohler. We thank A. Singh and S. Kane for polyclonal antibody development, and W. Colpoys, L. Cunningham, J. Simendinger, K. Crosby and G. Innocenti for help with western blot analysis, immunohistochemistry, immunofluorescence and flow cytometry. We thank K. Smith for help with polyclonal purification and M. Lewis for help with animal immunization and spleen isolation. We thank C. Reeves for help with DNA sequencing and J. Knott for peptide antigen synthesis. Finally, we thank P. Hornbeck, C. Hoffman, S. Chow and T. Singleton for reading the manuscript and providing useful discussions.

Author information

Authors and Affiliations

Authors

Contributions

W.C.C., S.A.B. and R.D.P. developed the methodology, designed experiments, analyzed the data and wrote the manuscript. W.C.C. and S.A.B. performed experiments and did the bioinformatic analysis. S.S. designed experiments, analyzed data and wrote the manuscript. X.Z., S.M.S., J.S.W., J.G.B., R.K.R. and L.P. performed experiments. M.J.C. and J.R. helped analyze the data and write the manuscript.

Corresponding author

Correspondence to Roberto D Polakiewicz.

Ethics declarations

Competing interests

All authors are employees of Cell Signaling Technology.

Supplementary information

Supplementary Text and Figures

Supplementary Tables 1–3 and Supplementary Figures 1–8 (PDF 2216 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Cheung, W., Beausoleil, S., Zhang, X. et al. A proteomics approach for the identification and cloning of monoclonal antibodies from serum. Nat Biotechnol 30, 447–452 (2012). https://doi.org/10.1038/nbt.2167

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt.2167

This article is cited by

Search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research