Skip to main content
SpringerLink
Log in

Facile Affinity Maturation of Single-Domain Antibodies Using Next-Generation DNA Sequencing

  • Protocol
  • First Online:
Single-Domain Antibodies

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2446))

Abstract

Binding affinity is one of the primary determinants of antibody function. Here, we provide a protocol for simple and rapid affinity maturation of single-domain antibodies (sdAbs) using tandem phage display selection and next-generation DNA sequencing. The sequence of a model camelid sdAb directed against Clostridioides difficile toxin A (A26.8) was diversified using either random or site-saturation mutagenesis and cloned into a phagemid vector upstream of gene 3. The resulting phage-displayed sdAb libraries were panned against C. difficile toxin A and the panning outputs interrogated using Illumina MiSeq sequencing. Through bioinformatic analyses, we were able to identify individual affinity-enhancing amino acid substitutions in the sdAb complementarity-determining regions that, when combined, resulted in affinity improvements of approximately 10-fold. The advantages of this method are that it does not require extensive screening and characterization of individual clones, nor structural information on the mechanism of the sdAb:antigen interaction.

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

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 169.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 219.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 299.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Muyldermans S (2013) Nanobodies: natural single-domain antibodies. Annu Rev Biochem 82:775–797

    Article  CAS  Google Scholar 

  2. Zavrtanik U, Lukan J, Loris R et al (2018) Structural basis of epitope recognition by heavy-chain camelid antibodies. J Mol Biol 430:4369–4386

    Article  CAS  Google Scholar 

  3. Brooks CL, Rossotti MA, Henry KA (2018) Immunological functions and evolutionary emergence of heavy-chain antibodies. Trends Immunol 39:956–996

    Google Scholar 

  4. Henry KA, MacKenzie CR (2018) Antigen recognition by single-domain antibodies: structural latitudes and constraints. MAbs 10:815–826

    Article  CAS  Google Scholar 

  5. Gram H, Marconi LA, Barbas CF 3rd, et al (1992) In vitro selection and affinity maturation of antibodies from a naïve combinatorial immunoglobulin library. Proc Natl Acad Sci U S A 89:3576–3580

    Google Scholar 

  6. 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  Google Scholar 

  7. Peltomaa R, Benito-Peña E, Barderas R et al (2019) Phage display in the quest for new selective recognition elements for biosensors. ACS Omega 4:11569–11580

    Article  CAS  Google Scholar 

  8. Kuroda D, Shirai H, Jacobson MP et al (2012) Computer-aided antibody design. Protein Eng Des Sel 25:507–521

    Article  CAS  Google Scholar 

  9. Hussack G, Arbabi-Ghahroudi M, van Faassen H et al (2011) Neutralization of Clostridium difficile toxin A with single-domain antibodies targeting the cell receptor binding domain. J Biol Chem 286:8961–8976

    Article  CAS  Google Scholar 

  10. Andrews S (2010) FastQC: a quality control tool for high throughput sequence data. http://www.bioinformatics.babraham.ac.uk/projects/fastqc

  11. Magoc T, Salzberg SL (2011) FLASH: fast length adjustment of short reads to improve genome assemblies. Bioinformatics 27:2957–2963

    Article  CAS  Google Scholar 

  12. Schmieder R, Edwards R (2011) Quality control and preprocessing of metagenomic datasets. Bioinformatics 27:863–864

    Article  CAS  Google Scholar 

  13. Cadwell RC, Joyce GF (1992) Randomization of genes by PCR mutagenesis. PCR Methods Appl 2:28–33

    Article  CAS  Google Scholar 

  14. Papworth C, Bauer J, Braman J, Wright D (1996) Site-directed mutagenesis in one day with >80% efficiency. Strategies 9:3–4

    Article  Google Scholar 

  15. Baral TN, MacKenzie R, Arbabi Ghahroudi M (2013) Single-domain antibodies and their utility. Curr Protoc Immunol 103:Unit 2.17

    Google Scholar 

  16. Hussack G, Baral TN, Baardsnes J et al (2017) A novel affinity tag, ABTAG, and its application to the affinity screening of single-domain antibodies selected by phage display. Frontiers Immunol 8:14

    Google Scholar 

  17. Henry KA (2018) Next-generation DNA sequencing of VH/VL repertoires: a primer and guide to applications in single-domain antibody discovery. Methods Mol Biol 1701:425–446

    Article  CAS  Google Scholar 

Download references

Acknowledgments

We gratefully acknowledge the excellent technical assistance of Qingling Yang, Hiba Kandalaft, and Gabrielle Richard. This work was supported by funding from the National Research Council Canada.

Conflict of Interest

The authors declare that they have no conflicts of interest.

Author information

Authors and Affiliations

  1. Human Health Therapeutics Research Centre, Life Sciences Division, National Research Council Canada, Ottawa, ON, Canada

    Michael J. Lowden, Henk van Faassen, Shalini Raphael, Shannon Ryan, Greg Hussack & Kevin A. Henry

  2. Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, ON, Canada

    Kevin A. Henry

Authors
  1. Michael J. Lowden
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Henk van Faassen
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Shalini Raphael
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Shannon Ryan
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Greg Hussack
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Kevin A. Henry
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Kevin A. Henry .

Editor information

Editors and Affiliations

  1. Human Health Therapeutics Research Centre, National Research Council Canada, Ottawa, ON, Canada

    Greg Hussack

  2. Human Health Therapeutics Research Centre, National Research Council Canada, Ottawa, ON, Canada

    Kevin A. Henry

Rights and permissions

Reprints and permissions

Copyright information

© 2022 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

Lowden, M.J., van Faassen, H., Raphael, S., Ryan, S., Hussack, G., Henry, K.A. (2022). Facile Affinity Maturation of Single-Domain Antibodies Using Next-Generation DNA Sequencing. In: Hussack, G., Henry, K.A. (eds) Single-Domain Antibodies. Methods in Molecular Biology, vol 2446. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2075-5_12

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-2075-5_12

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2074-8

  • Online ISBN: 978-1-0716-2075-5

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation