Abstract
The application of phage display to the screening of antibody V-gene libraries promises to replace animal immunization and hybridoma technology as a means of antibody generation (1,2). Synthetic libraries offer the potential for expanding the immune system repertoire and designing libraries for particular sets of antigens. We have developed a method for the generation of synthetic antibody gene libraries by which spiked oligonucleotides are assembled by a ligase chain reaction protocol (3). This is a synthetic adaptation of a reaction that was previously used as a diagnostic tool (4,5).
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Marks, J. D., Hoogenboom, H. R., Bonnert, T. P., McCafferty, J., Griffiths, A. D., and Winter, G. (1991) By-passing immunization: human antibodies from V-gene libraries displayed on phage. J. Mol. Biol. 222, 581–597.
Griffiths, A. D., Malmqvist, M., Marks, J. D., Bye, J. M., Embleton, M. J., McCafferty, J., Baier, M., Holliger, K. P., Gorick, B. D., Hughes-Jones, N. C., Hoogenboom, H. R., and Winter, G. (1993) Human anti-self antibodies with high specificity from phage display libraries. EMBO J. 12, 725–734.
Deng, S.-J., MacKenzie, C. R., and Narang, S. A. (1993) Simultaneous randomization of antibody CDRs by a synthetic ligase chain reaction strategy. Nucleic Acids Res. 21, 4418–4419
Landegren, U., Kaiser, R., Sanders, J., and Hood, L. (1988) A ligase-mediated gene detection technique. Science 241, 1077–1080.
Barany, F. (1991) Genetic disease detection and DNA amplification using cloned thermostable ligase. Proc. Natl. Acad. Sci. USA 88, 189–193.
Bundle, D. R., Eichler, E., Gidney, M. A. J., Meldal, M., Ragauskas, A., Sigurskjold, B. W., Sinnott, B., Watson, D. C., Yaguchi, M., and Young, N. M. (1994) Molecular recognition of a Salmonella trisaccharide epitope by monoclonal antibody Se155-4. Biochemistry 33, 5172–5182.
Cygler, M., Rose, D. R., and Bundle, D. R. (1991) Recognition of a cell-surface oligosaccharide of pathogenic Salmonella by an antibody fragment Science 253, 442–445.
Anand, N. N., Mandal, S., MacKenzie, C. R., Sadowska, J., Sigurskjold, B., Young, N. M., Bundle, D. R., and Narang, S. A. (1991) Bacterial expression and secretion of various single-chain Fv genes encoding proteins specific for a Salmonella sero-type B O-antigen. J. Biol. Chem. 266, 21,874–21,879
Brummell, D. A., Sharma, V. P., Anand, N. N., Bilous, D., Dubuc, G., Michniewicz, J., MacKenzie, C. R., Sadowska, J., Sigurskjold, B. W., Sinnott, B., Young, N. M., Bundle, D. R., and Narang, S. A. (1993) Probing the combining site of an anti-carbohydrate antibody by saturation-mutagenesis: role of the heavy-chain CDR3 residues. Biochemistry 32, 1180–1187.
Deng, S.-J., MacKenzie, C. R., Sadowska, J., Michniewicz, J., Young, N. M., Bundle, D. R., and Narang, S. A. (1994) Selection of antibody single-chain variable fragments with improved carbohydrate-binding by phage display. J. Biol. Chem. 269, 9533–9538.
Schlom, J., Eggensperger, D., Colcher, D., Molinolo, A., Houchens, D., Miller, L. S., Hinkle, G., and Siler, K. (1992) Cancer Res. 53, 1067–1072.
Berek, C. and Milstein, C. (1987) Mutation drift and repertoire shift in the maturation of the immune response. Immunol. Rev. 96, 23–41.
Hutchison, C. A., Nordeen, S. K., Vogt, K., and Edgell, M. H. (1986) A complete library of point substitution mutants in the glucocorticoid response element of mouse mammary tumor virus. Proc. Natl. Acad. Sci. USA 83, 710–714.
Cwirla, S. E., Peters, E. H., Barrett, R. W., and Dower, W. S. (1990) Peptides on phage: a vast library of peptides for identifying ligands. Proc. Natl. Acad. Sci. USA 87, 6378–6382.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 1995 Humana Press Inc.
About this protocol
Cite this protocol
Deng, Sj., MacKenzie, C.R., Narang, S.A. (1995). Synthetic Antibody Gene Libraries for In Vitro Affinity Maturation. In: Paul, S. (eds) Antibody Engineering Protocols. Methods In Molecular Medicine™, vol 51. Humana Press. https://doi.org/10.1385/0-89603-275-2:329
Download citation
DOI: https://doi.org/10.1385/0-89603-275-2:329
Publisher Name: Humana Press
Print ISBN: 978-0-89603-275-0
Online ISBN: 978-1-59259-538-9
eBook Packages: Springer Protocols