Abstract
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.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Boder ET, Wittrup KD (1997) Yeast surface display for screening combinatorial polypeptide libraries. Nat Biotechnol 15:553–557
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 1319:51–63
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 7
Sheehan J, Marasco WA (2015) Phage and yeast display. Microbiol Spectr 3:AID-0028-2014
Van Deventer JA, Wittrup KD (2014) Yeast surface display for antibody isolation: library construction, library screening, and affinity maturation. Methods Mol Biol 1131:151–181
Chao G, Lau WL, Hackel BJ, Sazinsky SL, Lippow SM, Wittrup KD (2006) Isolating and engineering human antibodies using yeast surface display. Nat Protoc 1:755–768
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 21:163–170
Feldhaus MJ, Siegel RW (2004) Yeast display of antibody fragments: a discovery and characterization platform. J Immunol Methods 290:69–80
Perkins S, Foung SK (1995) Stabilizing antibody secretion of human Epstein Barr virus-activated B-lymphocytes with hybridoma formation by electrofusion. Methods Mol Biol 48:295–307
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:e1002653
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 85:10451–10463
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 74:10407–10416
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 4:372–384
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 351:158–169
Acknowledgments
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.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2019 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Keck, Zy., Wang, Y., Lau, P., Foung, S.K.H. (2019). Isolation of HCV Neutralizing Antibodies by Yeast Display. In: Law, M. (eds) Hepatitis C Virus Protocols . Methods in Molecular Biology, vol 1911. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8976-8_27
Download citation
DOI: https://doi.org/10.1007/978-1-4939-8976-8_27
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-8975-1
Online ISBN: 978-1-4939-8976-8
eBook Packages: Springer Protocols