Skip to main content
SpringerLink
Log in

Magnetic Nanoparticle-Based Semi-Automated Panning for High-Throughput Antibody Selection

  • Protocol
  • First Online:
Phage Display

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1701))

Abstract

The application of recombinant human antibodies is growing rapidly mainly in the field of diagnostics and therapeutics. To identify antibodies against a specific antigen, panning selection is carried out using different display technologies. Phage display technology remains the preferred platform due to its robustness and efficiency in biopanning experiments. There are both manual and semi-automated panning selections using polystyrene plastic, magnetic beads, and nitrocellulose as the immobilizing solid surface. Magnetic nanoparticles allow for improved antigen binding due to their large surface area. The Kingfisher Flex magnetic particle processing system was originally designed to aid in RNA, DNA, and protein extraction using magnetic beads. However, the system can be programmed for antibody phage display panning. The automation allows for a reduction in human error and improves reproducibility in between selections with the preprogrammed movements. The system requires minimum human intervention to operate; however, human intervention is needed for post-panning steps like phage rescue. In addition, polyclonal and monoclonal ELISA can be performed using the semi-automated platform to evaluate the selected antibody clones. This chapter will summarize the suggested protocol from the panning stage till the monoclonal ELISA evaluation. Other than this, important notes on the possible optimization and troubleshooting are also included at the end of this chapter.

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 129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 169.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 279.99
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. Chin CF, Ler LW, Choong YS, Ong EBB, Ismail A, Tye GJ, Lim TS (2016) Application of streptavidin mass spectrometric immunoassay tips for immunoaffinity based antibody phage display panning. J Microbiol Methods 120:6–14. https://doi.org/10.1016/j.mimet.2015.11.007

    Article  CAS  PubMed  Google Scholar 

  2. Cahill DJ (2001) Protein and antibody arrays and their medical applications. J Immunol Methods 250(1):81–91

    Article  CAS  PubMed  Google Scholar 

  3. Marx U, Embleton MJ, Fischer R, Gruber FP, Hansson U, Heuer J, De Leeuw WA, Logtenberg T, Merz W, Portetelle D (1997) Monoclonal antibody production. ATLA Nottingham 25:121–138

    Google Scholar 

  4. Ma JK, Drake PM, Christou P (2003) The production of recombinant pharmaceutical proteins in plants. Nat Rev Genet 4(10):794–805

    Article  CAS  PubMed  Google Scholar 

  5. Azzazy HM, Highsmith WE (2002) Phage display technology: clinical applications and recent innovations. Clin Biochem 35(6):425–445

    Article  CAS  PubMed  Google Scholar 

  6. Hoogenboom HR (2002) Overview of antibody phage-display technology and its applications. Methods Mol Biol 178:1–37

    CAS  PubMed  Google Scholar 

  7. Kehoe JW, Kay BK (2005) Filamentous phage display in the new millennium. Chem Rev 105(11):4056–4072

    Article  CAS  PubMed  Google Scholar 

  8. Ling MM (2003) Large antibody display libraries for isolation of high-affinity antibodies. Comb Chem High Throughput Screen 6(5):421–432

    Article  CAS  PubMed  Google Scholar 

  9. Bahara NHH, Chin ST, Choong YS, Lim TS (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(1):35–43

    Article  CAS  Google Scholar 

  10. 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. https://doi.org/10.1016/j.toxicon.2016.04.032

    Article  CAS  PubMed  Google Scholar 

  11. 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(2, Part B):512–523. https://doi.org/10.1016/j.molimm.2015.07.040

    Article  CAS  PubMed  Google Scholar 

  12. Hanes J, Schaffitzel C, Knappik A, Plückthun A (2000) Picomolar affinity antibodies from a fully synthetic naive library selected and evolved by ribosome display. Nat Biotechnol 18(12):1287–1292

    Article  CAS  PubMed  Google Scholar 

  13. Rothe C, Urlinger S, Löhning C, Prassler J, Stark Y, Jäger U, Hubner B, Bardroff M, Pradel I, Boss M (2008) The human combinatorial antibody library HuCAL GOLD combines diversification of all six CDRs according to the natural immune system with a novel display method for efficient selection of high-affinity antibodies. J Mol Biol 376(4):1182–1200

    Article  CAS  PubMed  Google Scholar 

  14. Knappik A, Ge L, Honegger A, Pack P, Fischer M, Wellnhofer G, Hoess A, WoÈlle 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(1):57–86

    Article  CAS  PubMed  Google Scholar 

  15. Lee CV, Liang W-C, Dennis MS, Eigenbrot C, Sidhu SS, Fuh G (2004) High-affinity human antibodies from phage-displayed synthetic Fab libraries with a single framework scaffold. J Mol Biol 340(5):1073–1093

    Article  CAS  PubMed  Google Scholar 

  16. Sidhu SS, Li B, Chen Y, Fellouse FA, Eigenbrot C, Fuh G (2004) Phage-displayed antibody libraries of synthetic heavy chain complementarity determining regions. J Mol Biol 338(2):299–310

    Article  CAS  PubMed  Google Scholar 

  17. Strachan G, McElhiney J, Drever M, McIntosh F, Lawton L, Porter A (2002) Rapid selection of anti-hapten antibodies isolated from synthetic and semi-synthetic antibody phage display libraries expressed in Escherichia coli. FEMS Microbiol Lett 210(2):257–261

    Article  CAS  PubMed  Google Scholar 

  18. Lim BN, Tye GJ, Choong YS, Ong EBB, Ismail A, Lim TS (2014) Principles and application of antibody libraries for infectious diseases. Biotechnol Lett 36(12):2381–2392. https://doi.org/10.1007/s10529-014-1635-x

    Article  CAS  PubMed  Google Scholar 

  19. Winter G, Griffiths AD, Hawkins RE, Hoogenboom HR (1994) Making antibodies by phage display technology. Annu Rev Immunol 12(1):433–455. https://doi.org/10.1146/annurev.iy.12.040194.002245

    Article  CAS  PubMed  Google Scholar 

  20. Konthur Z, Wilde J, Lim TS (2010) Semi-automated magnetic bead-based antibody selection from phage display libraries. Antibody Eng 2010:267–287

    Article  Google Scholar 

  21. Hamilton S (2002) Introduction to screening automation. High throughput screening. Methods Mol Biol 190:169–189

    CAS  PubMed  Google Scholar 

  22. Kala M, Bajaj K, Sinha S (1997) Magnetic bead enzyme-linked immunosorbent assay (ELISA) detects antigen-specific binding by phage-displayed scFv antibodies that are not detected with conventional ELISA. Anal Biochem 254(2):263–266

    Article  CAS  PubMed  Google Scholar 

  23. Konthur Z, Walter G (2002) Automation of phage display for high-throughput antibody development. Targets 1(1):30–36

    Article  CAS  Google Scholar 

  24. Barbas CF, Kang AS, Lerner RA, Benkovic SJ (1991) Assembly of combinatorial antibody libraries on phage surfaces: the gene III site. Proc Natl Acad Sci 88(18):7978–7982

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Ward R, Clark M, Lees J, Hawkins N (1996) Retrieval of human antibodies from phage-display libraries using enzymatic cleavage. J Immunol Methods 189(1):73–82

    Article  CAS  PubMed  Google Scholar 

  26. Hallborn J, Carlsson R (2002) Automated screening procedure for high-throughput generation of antibody fragments. BioTechniques 33:30–37

    Google Scholar 

  27. Walter G, Konthur Z, Lehrach H (2001) High-throughput screening of surface displayed gene products. Comb Chem High Throughput Screen 4(2):193–205. https://doi.org/10.2174/1386207013331228

    Article  CAS  PubMed  Google Scholar 

  28. Liu B, Huang L, Sihlbom C, Burlingame A, Marks JD (2002) Towards proteome-wide production of monoclonal antibody by phage display. J Mol Biol 315(5):1063–1073

    Article  CAS  PubMed  Google Scholar 

  29. Schwenk JM, Lindberg J, Sundberg M, Uhlén M, Nilsson P (2007) Determination of binding specificities in highly multiplexed bead-based assays for antibody proteomics. Mol Cell Proteomics 6(1):125–132

    Article  CAS  PubMed  Google Scholar 

  30. Behrens CR, Liu B (2014) Methods for site-specific drug conjugation to antibodies. MAbs 1:46–53

    Article  Google Scholar 

  31. Ta HT, Peter K, Hagemeyer CE (2012) Enzymatic antibody tagging: toward a universal biocompatible targeting tool. Trends Cardiovasc Med 22(4):105–111

    Article  CAS  PubMed  Google Scholar 

  32. Turunen L, Takkinen K, Söderlund H, Pulli T (2009) Automated panning and screening procedure on microplates for antibody generation from phage display libraries. J Biomol Screen. https://doi.org/10.1177/1087057108330113

Download references

Acknowledgment

The authors would like to acknowledge the support of USM Research University Individual grant (1001/CIPPM/812173) and Malaysian Ministry of Education through the Higher Institution Centre of Excellence (HICoE) Grant (Grant No: 311/CIPPM/44001005).

Author information

Authors and Affiliations

  1. Analytical Biochemistry Research Centre, Institute for Research in Molecular Medicine, Universiti Sains Malaysia, 11800, Minden, Penang, Malaysia

    Angela Chiew Wen Ch’ng & Nurul Hamizah Binti Hamidon

  2. Max Planck Institute of Colloids and Interfaces, Mühlenberg 1, 14476, Potsdam, Germany

    Zoltán Konthur

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

    Theam Soon Lim

  4. Analytical Biochemistry Research Centre, Universiti Sains Malaysia, Minden, Penang, Malaysia

    Theam Soon Lim

Authors
  1. Angela Chiew Wen Ch’ng
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Nurul Hamizah Binti Hamidon
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Zoltán Konthur
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. 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. Technische Universität Braunschweig, Braunschweig, Germany

    Michael Hust

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

    Theam Soon Lim

Rights and permissions

Reprints and permissions

Copyright information

© 2018 Springer Science+Business Media LLC

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Ch’ng, A.C.W., Hamidon, N.H.B., Konthur, Z., Lim, T.S. (2018). Magnetic Nanoparticle-Based Semi-Automated Panning for High-Throughput Antibody Selection. 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_16

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-7447-4_16

  • 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

Publish with us

Policies and ethics

Search

Navigation