SpringerLink
Log in

Antibody Phage Display

  • Protocol
  • First Online:
Phage Display

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2702))

Abstract

The application of antibodies has transcended across many areas of work but mainly as a research tool, for diagnostic and for therapeutic applications. Antibodies are immunoproteins from vertebrates that have the unique property of specifically binding foreign molecules and distinguish target antigens. This property allows antibodies to effectively protect the host from infections. Apart from the hybridoma technology using transgenic animals, antibody phage display is commonly considered the gold standard technique for the isolation of human monoclonal antibodies. The concept of antibody phage display surrounds the ability to display antibody fragments on the surface of M13 bacteriophage particles with the corresponding gene packaged within the particle. A repetitive in vitro affinity based selection process permits the enrichment of target specific binders. This process of recombinant human monoclonal antibody generation also enables additional engineering for various applications. This makes phage display an indispensable technique for antibody development and engineering activities.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution

Subscribe and save

Springer+ Basic
EUR 32.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or Ebook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Protocol
EUR 44.95
Price includes VAT (Thailand)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
EUR 234.33
Price includes VAT (Thailand)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Hardcover Book
EUR 279.99
Price excludes VAT (Thailand)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free ship** worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Similar content being viewed by others

References

  1. Hoffman W, Lakkis FG, Chalasani G et al (2016) B cells, antibodies, and more. Clin J Am Soc Nephrol 11:137 –154

    Article  CAS  PubMed  Google Scholar 

  2. Chi X, Li Y, Qiu X et al (2020) V(D)J recombination, somatic hypermutation and class switch recombination of immunoglobulins: mechanism and regulation. Immunology 160:233 –247

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Lu RM, Hwang YC, Liu IJ, Lee CC, Tsai HZ, Li HJ, Wu HC et al (2020) Development of therapeutic antibodies for the treatment of diseases. J Biomed Sci 27(1):1–30

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  PubMed  Google Scholar 

  5. Barbas CF, Kang AS, Lerner RA, Benkovic SJ et al (1991) Assembly of combinatorial antibody libraries on phage surfaces: the gene III site. Proc Natl Acad Sci U S A 88:7978

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Breitling F, Dübel S, Seehaus T, Klewinghaus I, Little M et al (1991) A surface expression vector for antibody screening. Gene 104:147 –153

    Article  CAS  PubMed  Google Scholar 

  7. McCafferty J, Griffiths AD, Winter G, Chiswell DJ et al (1990) Phage antibodies: filamentous phage displaying antibody variable domains. Nature 348:552 –554

    Article  CAS  PubMed  Google Scholar 

  8. Smith GP (1985) Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 228:1315 –1317

    Article  CAS  PubMed  Google Scholar 

  9. Alfaleh MA, Alsaab HO, Mahmoud AB, Alkayyal AA, Jones ML, Mahler SM, Hashem AM et al (2020) Phage display derived monoclonal antibodies: from bench to bedside. Front Immunol 11:1986

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Frenzel A, Schirrmann T, Hust M et al (2016) Phage display-derived human antibodies in clinical development and therapy. MAbs 8:1177

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Wen J, Yuan K, Wen J, Yuan K et al (2021) Phage display technology, phage display system, antibody library, prospects and challenges. Adv Microbiol 11:181 –189

    Article  CAS  Google Scholar 

  12. Lim CC, Chan SK, Lim YY, Ishikawa Y, Choong YS, Nagaoka Y, Lim TS et al (2021) Development and structural characterisation of human scFv targeting MDM2 spliced variant MDM215kDa. Mol Immunol 135:191 –203

    Article  CAS  PubMed  Google Scholar 

  13. Russo G, Meier D, Helmsing S, Wenzel E, Oberle F, Frenzel A, Hust M et al (2018) Parallelized antibody selection in microtiter plates. Methods Mol Biol 1701:273 –284

    Article  CAS  PubMed  Google Scholar 

  14. Porter RR (1963) Chemical structure of γ-globulin and antibodies. Br Med Bull 19:197 –201

    Article  CAS  PubMed  Google Scholar 

  15. Alzari PM, Lascombe MB, Poljak RJ et al (1988) Three-dimensional structure of antibodies. Annu Rev Immunol 6:555 –580

    Article  CAS  PubMed  Google Scholar 

  16. Barbié V, Lefranc MP (1998) The human immunoglobulin kappa variable (IGKV) genes and joining (IGKJ) segments. Exp Clin Immunogenet 15:171 –183

    Article  PubMed  Google Scholar 

  17. Malcolm S, Barton P, Murphy C, Ferguson-Smith MA, Bentley DL, Rabbitts TH et al (1982) Localization of human immunoglobulin kappa light chain variable region genes to the short arm of chromosome 2 by in situ hybridization. Proc Natl Acad Sci U S A 79:4957 –4961

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Chiu ML, Goulet DR, Teplyakov A, Gilliland GL et al (2019) Antibody structure and function: the basis for engineering therapeutics. Antibodies (Basel). https://doi.org/10.3390/ANTIB8040055

  19. Hanson QM, Barb AW (2015) A perspective on the structure and receptor binding properties of immunoglobulin G Fc. Biochemistry 54:2931 –2942

    Article  CAS  PubMed  Google Scholar 

  20. Bruhns P, Jönsson F (2015) Mouse and human FcR effector functions. Immunol Rev 268:25 –51

    Article  CAS  PubMed  Google Scholar 

  21. Power CA, Bates A (2019) David vs. Goliath: the structure, function, and clinical prospects of antibody fragments. Antibodies. https://doi.org/10.3390/ANTIB8020028

  22. Kinman AWL, Pompano RR (2019) Optimization of enzymatic antibody fragmentation for yield, efficiency, and binding affinity. Bioconjug Chem 30:800 –807

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Huston JS, Levinson D, Mudgett-Hunter M et al (1988) Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli. Proc Natl Acad Sci 85:5879 –5883

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Almagro JC, Pedraza-Escalona M, Arrieta HI, Pérez-Tapia SM et al (2019) Phage display libraries for antibody therapeutic discovery and development. Antibodies (Basel). https://doi.org/10.3390/ANTIB8030044

  25. Kafil V, Saei AA, Tohidkia MR, Barar J, Omidi Y et al (2020) Immunotargeting and therapy of cancer by advanced multivalence antibody scaffolds. J Drug Target 28:1018 –1033. https://doi.org/10.1080/1061186X20201772796

    Article  CAS  PubMed  Google Scholar 

  26. Steinwand M, Droste P, Frenzel A, Hust M, Dübel S, Schirrmann T et al (2014) The influence of antibody fragment format on phage display based affinity maturation of IgG. MAbs 6:204 –218

    Article  PubMed  Google Scholar 

  27. Omar N, Hamidon NH, Yunus MH, Noordin R, Choong YS, Lim TS et al (2018) Generation and selection of naïve Fab library for parasitic antigen: anti-BmSXP antibodies for lymphatic filariasis. Biotechnol Appl Biochem 65:346 –354

    Article  CAS  PubMed  Google Scholar 

  28. Loh Q, Leong SW, Tye GJ, Choong YS, Lim TS et al (2015) Improved Fab presentation on phage surface with the use of molecular chaperone coplasmid system. Anal Biochem 477:56 –61

    Article  CAS  PubMed  Google Scholar 

  29. Khodabakhsh F, Behdani M, Rami A, Kazemi-Lomedasht F et al (2018) Single-domain antibodies or nanobodies: a class of next-generation antibodies. Int Rev Immunol 37:316 –322

    Article  CAS  PubMed  Google Scholar 

  30. Muyldermans S, Atarhouch T, Saldanha J, Barbosa JARG, Hamers R et al (1994) Sequence and structure of VH domain from naturally occurring camel heavy chain immunoglobulins lacking light chains. Protein Eng 7:1129 –1135

    Article  CAS  PubMed  Google Scholar 

  31. Nuttall SD, Krishnan UV, Doughty L, Pearson K, Ryan MT, Hoogenraad NJ, Hattarki M, Carmichael JA, Irving RA, Hudson PJ et al (2003) Isolation and characterization of an IgNAR variable domain specific for the human mitochondrial translocase receptor Tom70. Eur J Biochem 270:3543 –3554

    Article  CAS  PubMed  Google Scholar 

  32. Hairul Bahara NH, Chin ST, Choong YS, Lim TS et al (2016) Construction of a semisynthetic human VH single-domain antibody library and selection of domain antibodies against α-crystalline of mycobacterium tuberculosis. J Biomol Screen 21:35 –43

    Article  PubMed  Google Scholar 

  33. Bélanger K, Tanha J (2021) High-efficacy, high-manufacturability human VH domain antibody therapeutics from transgenic sources. Protein Eng Des Sel 34:1 –7

    Article  Google Scholar 

  34. Rossotti MA, Bélanger K, Henry KA, Tanha J et al (2022) Immunogenicity and humanization of single-domain antibodies. FEBS J 289:4304 –4327

    Article  CAS  PubMed  Google Scholar 

  35. Ledsgaard L, Ljungars A, Rimbault C, Sørensen CV, Tulika T, Wade J, Wouters Y, McCafferty J, Laustsen AH et al (2022) Advances in antibody phage display technology. Drug Discov Today 27:2151 –2169

    Article  CAS  PubMed  Google Scholar 

  36. Nagano K, Tsutsumi Y (2021) Phage display technology as a powerful platform for antibody drug discovery. Viruses. https://doi.org/10.3390/V13020178

  37. Oreste U, Ametrano A, Coscia MR et al (2021) On origin and evolution of the antibody molecule. Biology (Basel) 10:1 –18

    Google Scholar 

  38. Hust M, Dübel S (2005) Phage display vectors for the in vitro generation of human antibody fragments. Methods Mol Biol 295:71 –96

    CAS  PubMed  Google Scholar 

  39. Hamidon NH, Suraiya S, Sarmiento ME, Acosta A, Norazmi MN, Lim TS (2018) Immune TB antibody phage display library as a tool to study B cell immunity in TB infections. Appl Biochem Biotechnol 184:852 –868

    Article  CAS  PubMed  Google Scholar 

  40. Rahumatullah A, Ahmad A, Noordin R, Lim TS (2015) Delineation of BmSXP antibody V-gene usage from a lymphatic filariasis based immune scFv antibody library. Mol Immunol 67:512 –523

    Article  CAS  PubMed  Google Scholar 

  41. Rahumatullah A, Karim IZA, Noordin R, Lim TS (2017) Antibody-based protective immunity against helminth infections: antibody phage display derived antibodies against BmR1 antigen. Int J Mol Sci. https://doi.org/10.3390/IJMS18112376

  42. Chan SK, Lim TS (2017) Immune human antibody libraries for infectious diseases. Adv Exp Med Biol 1053:61 –78

    Article  CAS  PubMed  Google Scholar 

  43. Wenzel EV, Bosnak M, Tierney R, Schubert M, Brown J, Dübel S, Efstratiou A, Sesardic D, Stickings P, Hust M (2020) Human antibodies neutralizing diphtheria toxin in vitro and in vivo. Sci Rep 10:1 –21

    Article  Google Scholar 

  44. Lai JY, Lim TS (2020) Infectious disease antibodies for biomedical applications: a mini review of immune antibody phage library repertoire. Int J Biol Macromol 163:640

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Rahumatullah A, Ahmad A, Noordin R, Lai JY, Baharudeen Z, Lim TS (2020) Applicability of Brugia malayi immune antibody library for the isolation of a human recombinant monoclonal antibody to Echinococcus granulosus antigen B. Exp Parasitol. https://doi.org/10.1016/J.EXPPARA.2020.108029

  46. Rahumatullah A, Balachandra D, Noordin R, Baharudeen Z, Lim YY, Choong YS, Lim TS (2021) Broad specificity of immune helminth scFv library to identify monoclonal antibodies targeting Strongyloides. Sci Rep 11:2502

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Kügler J, Wilke S, Meier D et al (2015) Generation and analysis of the improved human HAL9/10 antibody phage display libraries. BMC Biotechnol. https://doi.org/10.1186/S12896-015-0125-0

  48. 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

    Article  CAS  PubMed  Google Scholar 

  49. Lim TS, Mollova S, Rubelt F, Sievert V, Dübel S, Lehrach H, Konthur Z (2010) V-gene amplification revisited – an optimised procedure for amplification of rearranged human antibody genes of different isotypes. Nat Biotechnol 27:108 –117

    CAS  Google Scholar 

  50. Kügler J, Tomszak F, Frenzel A, Hust M (2018) Construction of human immune and naive scFv libraries. Methods Mol Biol 1701:3 –24

    Article  PubMed  Google Scholar 

  51. Lim CC, Choong YS, Lim TS (2019) Cognizance of molecular methods for the generation of mutagenic phage display antibody libraries for affinity maturation. Int J Mol Sci 20:1861

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. de Wildt RMT, Mundy CR, Gorick BD, Tomlinson IM (2000) Antibody arrays for high-throughput screening of antibody-antigen interactions. Nat Biotechnol 18:989 –994

    Article  PubMed  Google Scholar 

  53. Hayashi N, Welschof M, Zewe M, Braunagel M, Dubel S, Breitling F, Little M (1994) Simultaneous mutagenesis of antibody CDR regions by overlap extension and PCR. Biotechniques 17:310 , 312, 314–5

    Google Scholar 

  54. Erasmus MF, D’Angelo S, Ferrara F, Naranjo L, Teixeira AA, Buonpane R, Stewart SM, Nastri HG, Bradbury ARM (2021) A single donor is sufficient to produce a highly functional in vitro antibody library. Commun Biol 4:1 –16

    Article  Google Scholar 

  55. Knappik A, Ge L, Honegger A, Pack P, Fischer M, Wellnhofer G, Hoess A, Wölle J, Plückthun A, Virnekäs B (2000) Fully synthetic human combinatorial antibody libraries (HuCAL) based on modular consensus frameworks and CDRs randomized with trinucleotides. J Mol Biol 296:57 –86

    Article  CAS  PubMed  Google Scholar 

  56. Kumar R, Parray HA, Shrivastava T, Sinha S, Luthra K (2019) Phage display antibody libraries: a robust approach for generation of recombinant human monoclonal antibodies. Int J Biol Macromol 135:907 –918

    Article  CAS  PubMed  Google Scholar 

  57. Tiller T, Schuster I, Deppe D et al (2013) A fully synthetic human Fab antibody library based on fixed VH/VL framework pairings with favorable biophysical properties. MAbs 5:445

    Article  PubMed  PubMed Central  Google Scholar 

  58. Zadeh AS, Grässer A, Dinter H, Hermes M, Schindowski K (2019) Efficient construction and effective screening of synthetic domain antibody libraries. Methods Protoc 2:1 –19

    Google Scholar 

Download references

Acknowledgments

This work was supported by a Universiti Sains Malaysia, Special (Matching) Short-Term Grant with Project No: 304/CIPPM/6315708.

Author information

Authors and Affiliations

  1. Institute for Research in Molecular Medicine, Universiti Sains Malaysia, Penang, Malaysia

    Alia Nur, **g Yi Lai, Yee Siew Choong & Theam Soon Lim

  2. Institut für Biochemie, Biotechnologie und Bioinformatik, Departments Biotechnology and Medical Biotechnology, Technische Universität Braunschweig, Braunschweig, Germany

    Maren Schubert & Michael Hust

  3. School of Medical Sciences, Department of Medicine, Universiti Sains Malaysia, Kubang Kerian, Kelantan, Malaysia

    Wan Yus Haniff Wan Isa

  4. Analytical Biochemistry Research Center, Universiti Sains Malaysia, Penang, Malaysia

    Theam Soon Lim

Authors
  1. Alia Nur
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Maren Schubert
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. **g Yi Lai
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Michael Hust
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Yee Siew Choong
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Wan Yus Haniff Wan Isa
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Theam Soon Lim
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Theam Soon Lim .

Editor information

Editors and Affiliations

  1. Institut für Biochemie, Biotechnologie und Bioinformatik, Technische Universität Braunschweig, Braunschweig, Germany

    Michael Hust

  2. Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, Penang, Malaysia

    Theam Soon Lim

Rights and permissions

Reprints and permissions

Copyright information

© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Nur, A. et al. (2023). Antibody Phage Display. In: Hust, M., Lim, T.S. (eds) Phage Display. Methods in Molecular Biology, vol 2702. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3381-6_1

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-3381-6_1

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3380-9

  • Online ISBN: 978-1-0716-3381-6

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation