Abstract
With the identification of vast numbers of novel proteins through genomic and proteomic initiatives, the need for efficient processes to characterize and target them has increased. Antibodies are naturally designed molecules that can fulfill this need, and in vitro methodologies for isolating them from either immune or naïve sources have been extensively developed. However, access to pure protein antigens for screening purposes is a major hurdle due to the limitations associated with recombinant production of eukaryotic proteins. Consequently, rational peptide design based on proteomic methodologies such as protein modeling, secondary sequence prediction, and hydrophobicity/hydrophilicity prediction, in combination with other bioinformatics data, is being explored as a viable solution to isolate specific antibodies against difficult antigens. Single-domain antibodies are becoming the ideal antibody format due to their structural advantages and ease of production compared to conventional antibodies and antibody fragments derived from conventional antibodies. For screening purposes, phage display technology is a well-established technique. With this technique, a repertoire of antibody fragments can be displayed on the surface of filamentous phages (f1, fd, M13) followed by screening against various antigenic targets. Furthermore, the technique can be expanded to a high-throughput scale using a magnetic-based, in-solution panning protocol which allows for the screening of multiple target antigens simultaneously. In this chapter, we describe a semiautomated panning method to screen a naïve Camelidae library against rationally designed peptide antigens, followed by preliminary characterization of isolated binders.
This is National Research Council Canada Publication number: 50019.
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References
Hamers-Casterman C et al (1993) Naturally occurring antibodies devoid of light chains. Nature 363:446–448
Arbabi Ghahroudi M et al (1997) Selection and identification of single domain antibody fragments from camel heavy-chain antibodies. FEBS Lett 414:521–526
Ward ES et al (1989) Binding activities of a repertoire of single immunoglobulin variable domains secreted from Escherichia coli. Nature 341:544–546
Muyldermans S (2001) Single domain camel antibodies: current status. J Biotechnol 74:277–302
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 et al (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
Kehoe JW, Kay BK (2005) Filamentous phage display in the new millennium. Chem Rev 105:4056–4072
Pande J, Szewczyk MM, Grover AK (2010) Phage display: concept, innovations, applications and future. Biotechnol Adv 28:849–858
Konthur Z, Wilde J, Lim TS (2010) Semi-automated magnetic bead-based antibody selection from phage display libraries. In: Kontermann R, Dubel S (eds) Antibody engineering, 2nd edn. Springer-Verlag, Berlin Heidelberg, pp 267–287
Turunen L et al (2009) Automated panning and screening procedure on microplates for antibody generation from phage display libraries. J Biomol Screen 14:282–293
Sambrook J, Russell D (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
Stone E et al (2007) The assembly of single domain antibodies into bispecific decavalent molecules. J Immunol Methods 318:88–94
Arbabi-Ghahroudi M, Tanha J, MacKenzie R (2009) Isolation of monoclonal antibody fragments from phage display libraries. Methods Mol Biol 502:341–364
Zacher AN et al (1980) A new filamentous phage cloning vector: fd-tet. Gene 9:127–140
Doyle PJ et al (2008) Cloning, expression, and characterization of a single-domain antibody fragment with affinity for 15-acetyl-deoxynivalenol. Mol Immunol 45:3703–3713
Zhang J et al (2009) Transient expression and purification of chimeric heavy chain antibodies. Protein Expr Purif 65:77–82
Zhang J, MacKenzie R, Durocher Y (2009) Production of chimeric heavy-chain antibodies. Methods Mol Biol 525:323–336
Lee CM et al (2007) Selection of human antibody fragments by phage display. Nat Protoc 2:3001–3008
Acknowledgments
We thank Shalini Katary and Hong Tong-Sevinc for preparing and reviewing the KingFisher protocol and Henk van Faassen for performing the SPR experiments. We also thank the NRC Genomics and Health Initiative and Sentinel, The Canadian Network for the Development and Use of Bioactive Paper, for financial support. The authors declare no competing interests.
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Kumaran, J., MacKenzie, C.R., Arbabi-Ghahroudi, M. (2012). Semiautomated Panning of Naive Camelidae Libraries and Selection of Single-Domain Antibodies Against Peptide Antigens. In: Saerens, D., Muyldermans, S. (eds) Single Domain Antibodies. Methods in Molecular Biology, vol 911. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-968-6_7
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DOI: https://doi.org/10.1007/978-1-61779-968-6_7
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