Skip to main content

Thank you for visiting 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.

High-level generation of polyclonal antibodies by genetic immunization


Antibodies are important tools for investigating the proteome, but current methods for producing them have become a rate-limiting step1. A primary obstacle in most methods for generating antibodies or antibody-like molecules is the requirement for at least microgram quantities of purified protein. We have developed a technology for producing antibodies using genetic immunization2. Genetic immunization–based antibody production offers several advantages, including high throughput3 and high specificity. Moreover, antibodies produced from genetically immunized animals are more likely to recognize the native protein2. Here we show that a genetic immunization–based system can be used to efficiently raise useful antibodies to a wide range of antigens. We accomplished this by linking the antigen gene to various elements that enhance antigenicity and by codelivering plasmids encoding genetic adjuvants. Our system, which was tested by immunizing mice with >130 antigens, has shown a final success rate of 84%.

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

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Get just this article for as long as you need it


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

Figure 1: Design of genetic immunization vector.
Figure 2: Antibody responses of mice immunized with pBQAP10-AAT.
Figure 3: Western blot analysis of natural extracts.


  1. Kodadek, T. Protein microarrays: prospects and problems. Chem. Biol. 8, 105–115 (2001).

    Article  CAS  Google Scholar 

  2. Tang, D.-C., DeVit, M. & Johnston, S.A. Genetic immunization is a simple method for eliciting an immune response. Nature 356, 152–154 (1992).

    Article  CAS  Google Scholar 

  3. Sykes, K.F. & Johnston, S.A. Linear expression elements: a rapid, in vivo, method to screen for gene functions. Nat. Biotechnol. 17, 355–359 (1999).

    Article  CAS  Google Scholar 

  4. Babiuk, L.A., van Drunen Littel-van den Hurk, S. & Babiuk, S.L. Immunization of animals: from DNA to the dinner plate. Vet. Immunol. Immunopathol. 72, 189–202 (1999).

    Article  CAS  Google Scholar 

  5. Svanholm, C., Bandholtz, L., Lobell, A. & Wigzell, H. Enhancement of antibody responses by DNA immunization using expression vectors mediating efficient antigen secretion. J. Immunol. Methods 228, 121–130 (1999).

    Article  CAS  Google Scholar 

  6. Li, Z. et al. Immunogenicity of DNA vaccines expressing tuberculosis proteins fused to tissue plasminogen activator signal sequences. Infect. Immun. 67, 4780–4786 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Hammond, C. & Helenius, A. Quality control in the secretory pathway. Curr. Opin. Cell Biol. 7, 523–529 (1995).

    Article  CAS  Google Scholar 

  8. Terskikh, A.V. et al. “Peptabody”: a new type of high-avidity binding protein. Proc. Natl. Acad. Sci. USA 94, 1663–1668 (1997).

    Article  CAS  Google Scholar 

  9. St. Clair, N., Shenoy, B., Jacob, L.D. & Margolin, A.L. Cross-linked protein crystals for vaccine delivery. Proc. Natl. Acad. Sci. USA 96, 9469–9474 (1999).

    Article  CAS  Google Scholar 

  10. Valenzuela, P., Medina, A., Rutter, W.J., Ammerer, G. & Hall, B.D. Synthesis and assembly of hepatitis B virus surface antigen particles in yeast. Nature 298, 347–350 (1982).

    Article  CAS  Google Scholar 

  11. Scheerlinck, J.-P.Y. Genetic adjuvants for DNA vaccines. Vaccine 19, 2647–2656 (2001).

    Article  CAS  Google Scholar 

  12. Antonysamy, M.A. & Thomson, A.W. Flt3 ligand (FL) and its influence on immune reactivity. Cytokine 12, 87–100 (2000).

    Article  CAS  Google Scholar 

  13. International Human Genome Sequencing Consortium. Initial sequencing and analysis of the human genome. Nature 409, 860–921 (2001).

  14. Andre, S. et al. Increased immune response elicited by DNA vaccination with a synthetic gp120 sequence with optimized codon usage. J. Virol. 72, 1497–1503 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Stratford, R. et al. Influence of codon usage on the immunogenicity of a DNA vaccine against tetanus. Vaccine 19, 810–815 (2001).

    Article  Google Scholar 

  16. Smith, C., Day, P.J.R. & Walker, M.R. Generation of cohesive ends on PCR products by UDG-mediated excision of dU, and application for cloning into restriction digest-linearized vectors. PCR Methods Appl. 2, 328–332 (1993).

    Article  CAS  Google Scholar 

  17. Zinkernagel, R.M. What is missing in immunology to understand immunity? Nat. Immunol. 1, 181–185 (2000).

    Article  CAS  Google Scholar 

  18. King, C.A. et al. DNA vaccines with single-chain Fv fused to fragment C of tetanus toxin induce protective immunity against lymphoma and myeloma. Nat. Med. 4, 1281–1286 (1998).

    Article  CAS  Google Scholar 

  19. Dalum, I., Jensen, M.R., Hindersson, P., Elsner, H.I. & Mouritsen, S. Breaking of B cell tolerance toward a highly conserved self protein. J. Immunol. 157, 4796–4804 (1996).

    CAS  PubMed  Google Scholar 

  20. Jameson, B.A. & Wolf, H. The antigenic index: a novel algorithm for predicting antigenic determinants. Comput. Appl. Biosci. 4, 181–186 (1988).

    CAS  PubMed  Google Scholar 

  21. Pizza, M. et al. Identification of vaccine candidates against serotype B Meningococcus by whole-genome sequencing. Science 287, 1816–1820 (2000).

    Article  CAS  Google Scholar 

  22. Field, C.M., Oegema, K., Zheng, Y., Mitchison, T.J. & Walczak, C.E. Purification of cytoskeletal proteins using peptide antibodies. Methods Enzymol. 298, 525–541 (1998).

    Article  CAS  Google Scholar 

  23. Braun, P. et al. Proteome-scale purification of human proteins from bacteria. Proc. Natl. Acad. Sci. USA 99, 2654–2659 (2002).

    Article  CAS  Google Scholar 

  24. Barry, M.A., Barry, M.E. & Johnston, S.A. Production of monoclonal antibodies by genetic immunization. Biotechniques 16, 616–619 (1994).

    CAS  PubMed  Google Scholar 

  25. Chowdhury, P.S., Viner, J.L., Beers, R. & Pastan, I. Isolation of a high-affinity stable single-chain Fv specific for mesothelin from DNA-immunized mice by phage display and construction of a recombinant immunotoxin with anti-tumor activity. Proc. Natl. Acad. Sci. USA 95, 669–674 (1998).

    Article  CAS  Google Scholar 

  26. Niwa, H., Yamamura, K. & Miyazaki, J. Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 108, 193–199 (1991).

    Article  CAS  Google Scholar 

  27. Chang, C. et al. The Gal4 activation domain binds sug2 protein, a proteosome component, in vivo and in vitro. J. Biol. Chem. 276, 30956–30963 (2001).

    Article  CAS  Google Scholar 

  28. Sykes, K.F. & Johnston, S.A. Genetic live vaccines mimic the antigenicity but not pathogenicity of live viruses. DNA Cell Biol. 18, 521–531 (1999).

    Article  CAS  Google Scholar 

  29. Stemmer, W.P.C., Crameri, A., Ha, K.D., Brennan, T.M. & Heyneker, H.L. Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides. Gene 164, 49–53 (1995).

    Article  CAS  Google Scholar 

Download references


We thank Ryan Anholt, Anwarul Ferdous, Kyle Meinert, Joey Nguyen, Vickie Seward, Rebecca Solis and Greg Urquhart for technical assistance, Mike McGuire, Bao-Xi Qu and Irene Rombel for helpful discussions and Rick Lyons for supplying the Mtb extracts. This work was supported by grants from the Programs for Genomic Applications (PGA) from the US National Heart, Lung, and Blood Institute to S.A.J. at the University of Texas Southwestern (U01HL66880), and from the Countermeasures Program from the University of Texas Austin.

Author information

Authors and Affiliations


Corresponding author

Correspondence to Ross S Chambers.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Chambers, R., Johnston, S. High-level generation of polyclonal antibodies by genetic immunization. Nat Biotechnol 21, 1088–1092 (2003).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing