Skip to main content
SpringerLink
Log in

Isolation of Monoclonal Antibody Fragments from Phage Display Libraries

  • Protocol
Book cover Bacteriophages

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 502))

Abstract

Techniques developed over the past 20 years for the display of foreign peptides and proteins on the surfaces of filamentous bacteriophages have been a major driving force in the rapid development of recombinant antibody technology in recent years. With phage display of antibodies as one of its key components, recombinant antibody technology has led to the development of an increasing number of therapeutic monoclonal antibodies. Antibody gene libraries are fused to a gene encoding a phage coat protein. Recombinant phage expressing the resulting antibody libraries in fusion with the coat protein are propagated in Escherichia coli. Phage displaying monoclonal antibodies with specificities for target antigens are isolated from the libraries by a process called panning. The genes encoding the desired antibodies selected from the libraries are packaged within the phage particles, linking genotype and phenotype. Here, we describe the application of this technology to the construction of a phage-displayed single-domain antibody (sdAb) library based on the heavy chain antibody repertoire of a llama, the panning of the library against a peptide antigen and the expression, purification, and characterization of sdAbs isolated by panning.

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 149.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 199.99
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. Cwirla, S. E., Peters, E. A., Barrett, R. W., & Dower, W. J. (1990). Peptides on phage: a vast library of peptides for identifying ligands. Proc. Natl. Acad. Sci. U. S. A 87, 6378–6382.

    Article  CAS  PubMed  Google Scholar 

  2. Scott, J. K. & Smith, G. P. (1990). Searching for peptide ligands with an epitope library. Science 249, 386–390.

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  4. Arbabi-Ghahroudi, M., Desmyter, A., Wyns, L., Hamers, R., & Muyldermans, S. (1997). Selection and identification of single domain antibody fragments from camel heavy-chain antibodies. FEBS Lett 414, 521–526.

    Article  CAS  PubMed  Google Scholar 

  5. Barbas, C. F., III, Kang, A. S., Lerner, R. A., & Benkovic, S. J. (1991). Assembly of combinatorial antibody libraries on phage surfaces: the gene III site. Proc. Natl. Acad. Sci. U. S. A 88, 7978–7982.

    Article  CAS  PubMed  Google Scholar 

  6. Bradbury, A. & Cattaneo, A. (1995). The use of phage display in neurobiology. Trends Neurosci. 18, 243–249.

    Article  CAS  PubMed  Google Scholar 

  7. Bradbury, A. (2003). scFvs and beyond. Drug Discov. Today 8, 737–739.

    Article  PubMed  Google Scholar 

  8. Breitling, F., Dubel, S., Seehaus, T., Klewinghaus, I., & Little, M. (1991). A surface expression vector for antibody screening. Gene 104, 147–153.

    Article  CAS  PubMed  Google Scholar 

  9. Clackson, T., Hoogenboom, H. R., Griffiths, A. D., & Winter, G. (1991). Making antibody fragments using phage display libraries. Nature 352, 624–628.

    Article  CAS  PubMed  Google Scholar 

  10. Davies, J. & Riechmann, L. (1996). Single antibody domains as small recognition units: design and in vitro antigen selection of camelized, human VH domains with improved protein stability. Protein Eng 9, 531–537.

    Article  CAS  PubMed  Google Scholar 

  11. Hoogenboom, H. R., Griffiths, A. D., Johnson, K. S., Chiswell, D. J., Hudson, P., & Winter, G. (1991). Multi-subunit proteins on the surface of filamentous phage: methodologies for displaying antibody (Fab) heavy and light chains. Nucleic Acids Res. 19, 4133–4137.

    Article  CAS  PubMed  Google Scholar 

  12. Hoogenboom, H. R., de Bruine, A. P., Hufton, S. E., Hoet, R. M., Arends, J. W., & Roovers, R. C. (1998). Antibody phage display technology and its applications. Immunotechnology. 4, 1–20.

    Article  CAS  PubMed  Google Scholar 

  13. Lowman, H. B. (1997). Bacteriophage display and discovery of peptide leads for drug development. Annu. Rev. Biophys. Biomol. Struct. 26, 401–424.

    Article  CAS  PubMed  Google Scholar 

  14. Marks, J. D., Hoogenboom, H. R., Bonnert, T. P., McCafferty, J., Griffiths, A. D., & Winter, G. (1991). By-passing immunization. Human antibodies from V-gene libraries displayed on phage. J Mol Biol 222, 581–597.

    Article  CAS  PubMed  Google Scholar 

  15. McCafferty, J., Griffiths, A. D., Winter, G., & Chiswell, D. J. (1990). Phage antibodies: filamentous phage displaying antibody variable domains. Nature 348, 552–554.

    Article  CAS  PubMed  Google Scholar 

  16. Tanha, J., Dubuc, G., Hirama, T., Narang, S. A., & MacKenzie, C. R. (2002). Selection by phage display of llama conventional V(H) fragments with heavy chain antibody V(H)H properties. J. Immunol. Methods 263, 97–109.

    Article  CAS  PubMed  Google Scholar 

  17. Marks, J. D. & Bradbury, A. (2004). Selection of human antibodies from phage display libraries. Methods Mol. Biol. 248, 161–176.

    CAS  PubMed  Google Scholar 

  18. Sblattero, D. & Bradbury, A. (2000). Exploiting recombination in single bacteria to make large phage antibody libraries. Nat. Biotechnol. 18, 75–80.

    Article  CAS  PubMed  Google Scholar 

  19. Winter, G., Griffiths, A. D., Hawkins, R. E., & Hoogenboom, H. R. (1994). Making antibodies by phage display technology. Annu. Rev Immunol 12, 433–455.

    Article  CAS  PubMed  Google Scholar 

  20. Felici, F., Castagnoli, L., Musacchio, A., Jappelli, R., & Cesareni, G. (1991). Selection of antibody ligands from a large library of oligopeptides expressed on a multivalent exposition vector. J Mol. Biol. 222, 301–310.

    Article  CAS  PubMed  Google Scholar 

  21. Kay, B. K., Adey, N. B., He, Y. S., Manfredi, J. P., Mataragnon, A. H., & Fowlkes, D. M. (1993). An M13 phage library displaying random 38-amino-acid peptides as a source of novel sequences with affinity to selected targets. Gene 128, 59–65.

    Article  CAS  PubMed  Google Scholar 

  22. Scott, J. K., Loganathan, D., Easley, R. B., Gong, X., & Goldstein, I. J. (1992). A family of concanavalin A-binding peptides from a hexapeptide epitope library. Proc. Natl. Acad. Sci. U. S. A 89, 5398–5402.

    Article  CAS  PubMed  Google Scholar 

  23. Burton, D. R., Barbas, C. F., III, Persson, M. A., Koenig, S., Chanock, R. M., & Lerner, R. A. (1991). A large array of human monoclonal antibodies to type 1 human immunodeficiency virus from combinatorial libraries of asymptomatic seropositive individuals. Proc. Natl. Acad. Sci. U. S. A 88, 10134–10137.

    Article  CAS  PubMed  Google Scholar 

  24. Gram, H., Marconi, L. A., Barbas, C. F., III, Collet, T. A., Lerner, R. A., & Kang, A. S. (1992). In vitro selection and affinity maturation of antibodies from a naive combinatorial immunoglobulin library. Proc. Natl. Acad. Sci. U. S. A 89, 3576–3580.

    Article  CAS  PubMed  Google Scholar 

  25. Griffiths, A. D. (1993). Production of human antibodies using bacteriophage. Curr. Opin. Immunol 5, 263–267.

    Article  CAS  PubMed  Google Scholar 

  26. Hoogenboom, H. R., Marks, J. D., Griffiths, A. D., & Winter, G. (1992). Building antibodies from their genes. Immunol. Rev. 130, 41–68.

    Article  CAS  PubMed  Google Scholar 

  27. Muruganandam, A., Tanha, J., Narang, S., & Stanimirovic, D. (2002). Selection of phage-displayed llama single-domain antibodies that transmigrate across human blood-brain barrier endothelium. FASEB J. 16, 240–242.

    CAS  PubMed  Google Scholar 

  28. Vaughan, T. J., Williams, A. J., Pritchard, K., Osbourn, J. K., Pope, A. R., Earnshaw, J. C., McCafferty, J., Hodits, R. A., Wilton, J., & Johnson, K. S. (1996). Human antibodies with sub-nanomolar affinities isolated from a large non-immunized phage display library. Nat. Biotechnol. 14, 309–314.

    Article  CAS  PubMed  Google Scholar 

  29. Knappik, A., Ge, L., Honegger, A., Pack, P., Fischer, M., Wellnhofer, G., Hoess, A., Wolle, J., Pluckthun, A., & Virnekas, 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 

  30. Krebs, B., Rauchenberger, R., Reiffert, S., Rothe, C., Tesar, M., Thomassen, E., Cao, M., Dreier, T., Fischer, D., Hoss, A., Inge, L., Knappik, A., Marget, M., Pack, P., Meng, X. Q., Schier, R., Sohlemann, P., Winter, J., Wolle, J., & Kretzschmar, T. (2001). High-throughput generation and engineering of recombinant human antibodies. J. Immunol. Methods 254, 67–84.

    Article  CAS  PubMed  Google Scholar 

  31. Tanha, J., Xu, P., Chen, Z. G., Ni, F., Kaplan, H., Narang, S. A., & MacKenzie, C. R. (2001). Optimal design features of camelized human single-domain antibody libraries. J. Biol. Chem 276, 24774–24780.

    Article  CAS  PubMed  Google Scholar 

  32. Hawkins, R. E., Russell, S. J., & Winter, G. (1992). Selection of phage antibodies by binding affinity. Mimicking affinity maturation. J Mol. Biol. 226, 889–896.

    Article  CAS  PubMed  Google Scholar 

  33. Lavoie, T. B., Drohan, W. N., & Smith-Gill, S. J. (1992). Experimental analysis by site-directed mutagenesis of somatic mutation effects on affinity and fine specificity in antibodies specific for lysozyme. J Immunol 148, 503–513.

    CAS  PubMed  Google Scholar 

  34. Arap, M. A. (2005). Phage display technology: applications and innovations. Genet. Mol. Biol. 28, 1–9. Ref Type: Journal

    Article  CAS  Google Scholar 

  35. Conrad, U. & Scheller, J. (2005). Considerations on antibody-phage display methodology. Comb. Chem High Throughput. Screen. 8, 117–126.

    Article  CAS  PubMed  Google Scholar 

  36. Kirsch, M., Zaman, M., Meier, D., Dubel, S., & Hust, M. (2005). Parameters affecting the display of antibodies on phage. J Immunol Methods 301, 173–185.

    Article  CAS  PubMed  Google Scholar 

  37. Griffiths, A. D., Williams, S. C., Hartley, O., Tomlinson, I. M., Waterhouse, P., Crosby, W. L., Kontermann, R. E., Jones, P. T., Low, N. M., Allison, T. J., & et al (1994). Isolation of high affinity human antibodies directly from large synthetic repertoires. EMBO J 13, 3245–3260.

    CAS  PubMed  Google Scholar 

  38. Sidhu, S. S., Li, B., Chen, Y., Fellouse, F. A., Eigenbrot, C., & Fuh, G. (2004). Phage-displayed antibody libraries of synthetic heavy chain complementarity determining regions. J Mol. Biol. 338, 299–310.

    Article  CAS  PubMed  Google Scholar 

  39. Bradbury, A., Persic, L., Werge, T., & Cattaneo, A. (1993). Use of living columns to select specific phage antibodies. Biotechnology (N. Y.) 11, 1565–1569.

    Article  CAS  Google Scholar 

  40. Mutuberria, R., Hoogenboom, H. R., van der, L. E., de Bruine, A. P., & Roovers, R. C. (1999). Model systems to study the parameters determining the success of phage antibody selections on complex antigens. J Immunol Methods 231, 65–81.

    Google Scholar 

  41. Cai, X. & Garen, A. (1995). Anti-melanoma antibodies from melanoma patients immunized with genetically modified autologous tumor cells: selection of specific antibodies from single-chain Fv fusion phage libraries. Proc. Natl. Acad. Sci. U. S. A 92, 6537–6541.

    Article  CAS  PubMed  Google Scholar 

  42. Palmer, D. B., George, A. J., & Ritter, M. A. (1997). Selection of antibodies to cell surface determinants on mouse thymic epithelial cells using a phage display library. Immunology 91, 473–478.

    Article  CAS  PubMed  Google Scholar 

  43. Becerril, B., Poul, M. A., & Marks, J. D. (1999). Toward selection of internalizing antibodies from phage libraries. Biochem. Biophys. Res. Commun. 255, 386–393.

    Article  CAS  PubMed  Google Scholar 

  44. Poul, M. A., Becerril, B., Nielsen, U. B., Morisson, P., & Marks, J. D. (2000). Selection of tumor-specific internalizing human antibodies from phage libraries. J Mol. Biol. 301, 1149–1161.

    Article  CAS  PubMed  Google Scholar 

  45. Baek, H., Suk, K. H., Kim, Y. H., & Cha, S. (2002). An improved helper phage system for efficient isolation of specific antibody molecules in phage display. Nucleic Acids Res. 30, e18.

    Article  PubMed  Google Scholar 

  46. Chames, P. & Baty, D. (2000). Antibody engineering and its applications in tumor targeting and intracellular immunization. FEMS Microbiol. Lett. 189, 1–8. Ref Type: Journal

    Article  CAS  PubMed  Google Scholar 

  47. Harrison, J. L., Williams, S. C., Winter, G., & Nissim, A. (1996). Screening of phage antibody libraries. Methods Enzymol. 267, 83–109.

    Article  CAS  PubMed  Google Scholar 

  48. Duenas, M., Malmborg, A. C., Casalvilla, R., Ohlin, M., & Borrebaeck, C. A. (1996). Selection of phage displayed antibodies based on kinetic constants. Mol. Immunol 33, 279–285.

    Article  CAS  PubMed  Google Scholar 

  49. Mancini, N., Carletti, S., Perotti, M., Canducci, F., Mammarella, M., Sampaolo, M., & Burioni, R. (2004). Phage display for the production of human monoclonal antibodies against human pathogens. New Microbiol. 27, 315–328

    CAS  PubMed  Google Scholar 

  50. Muyldermans, S., Cambillau, C., & Wyns, L. (2001). Recognition of antigens by single-domain antibody fragments: the superfluous luxury of paired domains. Trends Biochem. Sci. 26, 230–235.

    Article  CAS  PubMed  Google Scholar 

  51. Tanha, J., Muruganandam, A., & Stanimirovic, D. (2003). Phage Display Technology for Identifying Specific Antigens on Brain Endothelial Cells. Methods Mol. Med. 89, 435–450.

    CAS  PubMed  Google Scholar 

  52. Sambrook, J., Fritsch, E. F., & Maniatis, T. (1989). Molecular Cloning: A laboratory Manual, 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

    Google Scholar 

  53. Tung, W. L. & Chow, K. C. (1995). A modified medium for efficient electrotransformation of E. coli. Trends Genet. 11, 128–129.

    Article  CAS  PubMed  Google Scholar 

  54. Harmsen, M. M., Ruuls, R. C., Nijman, I. J., Niewold, T. A., Frenken, L. G. J., & de Geus, B. (2000). Llama heavy-chain V regions consist of at least four distinct subfamilies revealing novel sequence features. Mol. Immunol 37, 579–590.

    Article  CAS  PubMed  Google Scholar 

  55. Anand, N. N., Dubuc, G., Phipps, J., MacKenzie, C. R., Sadowska, J., Young, N. M., Bundle, D. R., & Narang, S. A. (1991). Synthesis and expression in Escherichia coli of cistronic DNA encoding an antibody fragment specific for a Salmonella serotype B O-antigen. Gene 100, 39–44.

    Article  CAS  PubMed  Google Scholar 

  56. MacKenzie, C. R., Sharma, V., Brummell, D., Bilous, D., Dubuc, G., Sadowska, J., Young, N. M., Bundle, D. R., & Narang, S. A. (1994). Effect of C lambda-C kappa domain switching on Fab activity and yield in Escherichia coli: synthesis and expression of genes encoding two anti-carbohydrate Fabs. Biotechnology N. Y. 12, 390–395.

    Article  CAS  PubMed  Google Scholar 

  57. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.

    Article  CAS  PubMed  Google Scholar 

  58. Pace, C. N., Vajdos, F., Fee, L., Grimsley, G., & Gray, T. (1995). How to measure and predict the molar absorption coefficient of a protein. Protein Sci. 4, 2411–2423.

    Article  CAS  PubMed  Google Scholar 

  59. Yau, K. Y., Dubuc, G., Li, S., Hirama, T., MacKenzie, C. R., Jermutus, L., Hall, J. C., & Tanha, J. (2005). Affinity maturation of a V(H)H by mutational hotspot randomization. J Immunol Methods 297, 213–224.

    Article  CAS  PubMed  Google Scholar 

  60. Spinelli, S., Frenken, L., Bourgeois, D., de Ron, L., Bos, W., Verrips, T., Anguille, C., Cambillau, C., & Tegoni, M. (1996). The crystal structure of a llama heavy chain variable domain. Nat. Struct. Biol. 3, 752–757.

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Institute for Biological Sciences, National Research Council, The Antibody Engineering Group, Ottawa, Ontario, Canada

    Mehdi Arbabi-Ghahroudi, Jamshid Tanha & Roger MacKenzie

Authors
  1. Mehdi Arbabi-Ghahroudi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jamshid Tanha
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Roger MacKenzie
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. University of Leicester, Leicester, UK

    Martha R.J. Clokie

  2. Public Health Agency of Canada, Guelph, Ontario, Canada

    Andrew M. Kropinski

Rights and permissions

Reprints and permissions

Copyright information

© 2009 Humana Press, a part of Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Arbabi-Ghahroudi, M., Tanha, J., MacKenzie, R. (2009). Isolation of Monoclonal Antibody Fragments from Phage Display Libraries. In: Clokie, M.R., Kropinski, A.M. (eds) Bacteriophages. Methods in Molecular Biology™, vol 502. Humana Press. https://doi.org/10.1007/978-1-60327-565-1_20

Download citation

  • DOI: https://doi.org/10.1007/978-1-60327-565-1_20

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-60327-564-4

  • Online ISBN: 978-1-60327-565-1

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation