Abstract
This protocol describes the processes involved in the generation of human antibody libraries in Fab format. The antibody repertoire is derived from peripheral blood mononucleocytes focusing on different immunoglobulin isotypes. A two-step cloning process was used to generate a diverse human Fab library for subsequent selection by phage display. The method can be applied for the generation of both naive and immune antibody libraries. The naive repertoire allows for the library to be applied for the generation of human monoclonal antibodies against a broad range of target antigens making it a useful resource for antibody generation. However, the immune repertoire will be focused against target antigens from a particular disease. The protocol will focus on the generation of the library including the panning process.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Smith GP (1985) Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 228(4705):1315–1317
Barbas CF, Burton DR, Scott JK, Sivrmann GJ (2004) Phage display. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
Soumillion P, Jespers L, Bouchet M, Marchand-Brynaert J (1994) Phage display of enzymes and in vitro selection for catalytic activity. Appl Biochem Biotechnol 47(2–3):175–189
Fujita S, Taki T, Taira K (2005) Selection of an active enzyme by phage display on the basis of the enzyme’s catalytic activity in vivo. Chembiochem 6(2):315–321
Zoller F, Haberkorn U, Mier W (2011) Miniproteins as phage display-scaffolds for clinical applications. Molecules 16(3):2467–2485
Uchiyama F, Tanaka Y, Minari Y, Tokui N (2005) Designing scaffolds of peptides for phage display libraries. J Biosci Bioeng 99(5):448–456
Schirrmann T, Hust M (2010) Construction of human antibody gene libraries and selection of antibodies by phage display. Methods Mol Biol 651:177–209
Lim BN, Tye GJ, Choong YS, Ong EB, Ismail A, Lim TS (2014) Principles and application of antibody libraries for infectious diseases. Biotechnol Lett 36(12):2381–2392
Bain B, Brazil M (2003) Adalimumab. Nat Rev Drug Discov 2(9):693–694
Bradbury A, Velappan N, Verzillo V, Ovecka M et al (2003) Antibodies in proteomics I: generating antibodies. Trends Biotechnol 21(6):275–281
Ponsel D, Neugebauer J, Ladetzki-Baehs K, Tissot K (2011) High affinity, developability and functional size: the holy grail of combinatorial antibody library generation. Molecules 16(5):3675–3700
Krebs B, Rauchenberger R, Reiffert S, Rothe C (2001) High-throughput generation and engineering of recombinant human antibodies. J Immunol Methods 254(1):67–84
Konthur Z, Walter G (2002) Automation of phage display for high-throughput antibody development. Targets 1(1):30–36
Janeway CA, Travels P, Walport M, Capra JD (1999) Immunobiology: the immune system in health and disease, vol 157. Current Biology Publications, New York
Kindt T, Goldsby R, Osborne B (2007) Kuby immunology, 6th edn. WH Freeman, New York
Lim BN, Chin CF, Choong YS, Ismail A, Lim TS (2016) Generation of a naïve human single chain variable fragment (scFv) library for the identification of monoclonal scFv against salmonella Typhi Hemolysin E antigen. Toxicon 117:94–101
Zhu ZD, Dimitrov S (2009) Construction of a large naive human phage-displayed fab library through one-step cloning. Therapeutic Antibodies 6:129–142
Rahumatullah A, Ahmad A, Rahmah N, Lim TS (2015) Delineation of BmSXP antibody V-gene usage from a lymphatic filariasis based immune scFv antibody library. Mol Immunol 67:512–523
Shen Y, Yang X, Dong N, Xie X et al (2007) Generation and selection of immunized fab phage display library against human B cell lymphoma. Cell Res 17(7):650–660
Nelson AL (2010) Antibody fragments: hope and hype. MAbs 2(1):77–83
Ward ES, Gussow D, Griffiths AD, Jones PT, Winter G (1989) Binding activities of a repertoire of single immunoglobulin variable domains secreted from Escherichia coli. Nature 341(6242):544–546
Röthlisberger D, Honegger A, Plückthun A (2005) Domain interactions in the fab fragment: a comparative evaluation of the single-chain Fv and fab format engineered with variable domains of different stability. J Mol Biol 347(4):773–789
Hust M, Dübel S (2004) Mating antibody phage display with proteomics. Trends Biotechnol 22(1):8–14
Shahsavarian MA, Le Minoux D, Matti KM, Kaveri S et al (2014) Exploitation of rolling circle amplification for the construction of large phage-display antibody libraries. J Immunol Methods 407:26–34
Kirsch M, Zaman M, Meier D, Dubel S, Hust M (2005) Parameters affecting the display of antibodies on phage. J Immunol Methods 301(1):173–185
Kipriyanov SM, Moldenhauer G, Little M (1997) High level production of soluble single chain antibodies in small-scale Escherichia coli cultures. J Immunol Methods 200(1–2):69–77
Giudicelli V, Chaume D, Lefranc MP (2004) IMGT/V-QUEST, an integrated software program for immunoglobulin and T cell receptor V-J and V-D-J rearrangement analysis. Nucleic Acids Res 32:435–440
Acknowledgments
g849 The authors would like to acknowledge the support of the Malaysian Ministry of Education through the Higher Institution Centre of Excellence (HICoE) Grant (Grant No. 311/CIPPM/4401005).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media LLC
About this protocol
Cite this protocol
Omar, N., Lim, T.S. (2018). Construction of Naive and Immune Human Fab Phage-Display Library. In: Hust, M., Lim, T. (eds) Phage Display. Methods in Molecular Biology, vol 1701. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7447-4_2
Download citation
DOI: https://doi.org/10.1007/978-1-4939-7447-4_2
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7446-7
Online ISBN: 978-1-4939-7447-4
eBook Packages: Springer Protocols