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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
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.
Scott, J. K. & Smith, G. P. (1990). Searching for peptide ligands with an epitope library. Science 249, 386–390.
Smith, G. P. (1985). Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 228, 1315–1317.
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.
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.
Bradbury, A. & Cattaneo, A. (1995). The use of phage display in neurobiology. Trends Neurosci. 18, 243–249.
Bradbury, A. (2003). scFvs and beyond. Drug Discov. Today 8, 737–739.
Breitling, F., Dubel, S., Seehaus, T., Klewinghaus, I., & Little, M. (1991). A surface expression vector for antibody screening. Gene 104, 147–153.
Clackson, T., Hoogenboom, H. R., Griffiths, A. D., & Winter, G. (1991). Making antibody fragments using phage display libraries. Nature 352, 624–628.
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.
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.
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.
Lowman, H. B. (1997). Bacteriophage display and discovery of peptide leads for drug development. Annu. Rev. Biophys. Biomol. Struct. 26, 401–424.
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.
McCafferty, J., Griffiths, A. D., Winter, G., & Chiswell, D. J. (1990). Phage antibodies: filamentous phage displaying antibody variable domains. Nature 348, 552–554.
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.
Marks, J. D. & Bradbury, A. (2004). Selection of human antibodies from phage display libraries. Methods Mol. Biol. 248, 161–176.
Sblattero, D. & Bradbury, A. (2000). Exploiting recombination in single bacteria to make large phage antibody libraries. Nat. Biotechnol. 18, 75–80.
Winter, G., Griffiths, A. D., Hawkins, R. E., & Hoogenboom, H. R. (1994). Making antibodies by phage display technology. Annu. Rev Immunol 12, 433–455.
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.
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.
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.
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.
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.
Griffiths, A. D. (1993). Production of human antibodies using bacteriophage. Curr. Opin. Immunol 5, 263–267.
Hoogenboom, H. R., Marks, J. D., Griffiths, A. D., & Winter, G. (1992). Building antibodies from their genes. Immunol. Rev. 130, 41–68.
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.
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.
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.
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.
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.
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.
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.
Arap, M. A. (2005). Phage display technology: applications and innovations. Genet. Mol. Biol. 28, 1–9. Ref Type: Journal
Conrad, U. & Scheller, J. (2005). Considerations on antibody-phage display methodology. Comb. Chem High Throughput. Screen. 8, 117–126.
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.
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.
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.
Bradbury, A., Persic, L., Werge, T., & Cattaneo, A. (1993). Use of living columns to select specific phage antibodies. Biotechnology (N. Y.) 11, 1565–1569.
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.
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.
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.
Becerril, B., Poul, M. A., & Marks, J. D. (1999). Toward selection of internalizing antibodies from phage libraries. Biochem. Biophys. Res. Commun. 255, 386–393.
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.
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.
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
Harrison, J. L., Williams, S. C., Winter, G., & Nissim, A. (1996). Screening of phage antibody libraries. Methods Enzymol. 267, 83–109.
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.
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
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.
Tanha, J., Muruganandam, A., & Stanimirovic, D. (2003). Phage Display Technology for Identifying Specific Antigens on Brain Endothelial Cells. Methods Mol. Med. 89, 435–450.
Sambrook, J., Fritsch, E. F., & Maniatis, T. (1989). Molecular Cloning: A laboratory Manual, 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
Tung, W. L. & Chow, K. C. (1995). A modified medium for efficient electrotransformation of E. coli. Trends Genet. 11, 128–129.
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.
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.
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.
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.
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.
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.
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.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights 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