Isolation of HCV Neutralizing Antibodies by Yeast Display

  • Zhen-yong Keck
  • Yong Wang
  • Patrick Lau
  • Steven K. H. FoungEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1911)


Yeast surface display (YSD) enables efficient screening and selection of single chain variable fragments (scFvs) of heavy (VH) and light (VL) chains that bind to target antigen with different affinities. Assembly of a scFv library from cDNA usually involves adding different primers and linkers (Gly4/Ser)3 through multiple rounds of PCR amplification and purification. We describe here a simplified scFv assembly method by creating a modified YSD vector with a built-in linker that reduces the time of assembly and decreases accumulated base exchanges due to PCR errors. In addition, we describe a bias screening strategy toward maximizing novel antibodies of interest by a combination of memory B cell selection and depletion by binding to mutant antigens that do not bind to previously identified monoclonal antibodies.

Key words

Yeast surface display Human monoclonal antibody isolation Hepatitis C virus Virus neutralization Mutant antigen selection 



This work was support in part by National Institute of Allergy and Infectious Diseases/NIH grants R41-AI108024, U19-AI123862, R01-AI132213 and R21-AI126582 to SKHF.


  1. 1.
    Boder ET, Wittrup KD (1997) Yeast surface display for screening combinatorial polypeptide libraries. Nat Biotechnol 15:553–557CrossRefGoogle Scholar
  2. 2.
    Bidlingmaier S, Su Y, Liu B (2015) Combining phage and yeast cell surface antibody display to identify novel cell type-selective internalizing human monoclonal antibodies. Methods Mol Biol 1319:51–63CrossRefGoogle Scholar
  3. 3.
    Miller KD, Pefaur NB, Baird CL (2008) Construction and screening of antigen targeted immune yeast surface display antibody libraries. Curr Protoc Cytom Chapter 4:Unit 4 7Google Scholar
  4. 4.
    Sheehan J, Marasco WA (2015) Phage and yeast display. Microbiol Spectr 3:AID-0028-2014Google Scholar
  5. 5.
    Van Deventer JA, Wittrup KD (2014) Yeast surface display for antibody isolation: library construction, library screening, and affinity maturation. Methods Mol Biol 1131:151–181CrossRefGoogle Scholar
  6. 6.
    Chao G, Lau WL, Hackel BJ, Sazinsky SL, Lippow SM, Wittrup KD (2006) Isolating and engineering human antibodies using yeast surface display. Nat Protoc 1:755–768CrossRefGoogle Scholar
  7. 7.
    Feldhaus MJ, Siegel RW, Opresko LK, Coleman JR, Feldhaus JM, Yeung YA et al (2003) Flow-cytometric isolation of human antibodies from a nonimmune Saccharomyces cerevisiae surface display library. Nat Biotechnol 21:163–170CrossRefGoogle Scholar
  8. 8.
    Feldhaus MJ, Siegel RW (2004) Yeast display of antibody fragments: a discovery and characterization platform. J Immunol Methods 290:69–80CrossRefGoogle Scholar
  9. 9.
    Perkins S, Foung SK (1995) Stabilizing antibody secretion of human Epstein Barr virus-activated B-lymphocytes with hybridoma formation by electrofusion. Methods Mol Biol 48:295–307PubMedGoogle Scholar
  10. 10.
    Keck ZY, Xia J, Wang Y, Wang W, Krey T, Prentoe J et al (2012) Human monoclonal antibodies to a novel cluster of conformational epitopes on HCV E2 with resistance to neutralization escape in a genotype 2a isolate. PLoS Pathog 8:e1002653CrossRefGoogle Scholar
  11. 11.
    Keck ZY, Saha A, Xia J, Wang Y, Lau P, Krey T et al (2011) Mapping a region of hepatitis C virus E2 that is responsible for escape from neutralizing antibodies and a core CD81-binding region that does not tolerate neutralization escape mutations. J Virol 85:10451–10463CrossRefGoogle Scholar
  12. 12.
    Hadlock KG, Lanford RE, Perkins S, Rowe J, Yang Q, Levy S et al (2000) Human monoclonal antibodies that inhibit binding of hepatitis C virus E2 protein to CD81 and recognize conserved conformational epitopes. J Virol 74:10407–10416CrossRefGoogle Scholar
  13. 13.
    Smith K, Garman L, Wrammert J, Zheng NY, Capra JD, Ahmed R et al (2009) Rapid generation of fully human monoclonal antibodies specific to a vaccinating antigen. Nat Protoc 4:372–384CrossRefGoogle Scholar
  14. 14.
    Razai A, Garcia-Rodriguez C, Lou J, Geren IN, Forsyth CM, Robles Y et al (2005) Molecular evolution of antibody affinity for sensitive detection of botulinum neurotoxin type A. J Mol Biol 351:158–169CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Zhen-yong Keck
    • 1
  • Yong Wang
    • 1
  • Patrick Lau
    • 1
  • Steven K. H. Foung
    • 1
    Email author
  1. 1.Department of PathologyStanford University School of MedicineStanfordUSA

Personalised recommendations