Abstract
This protocol describes a method for creation of a highly diverse and functional synthetic phage-displayed repertoire of fully human domain antibodies (dAbs). The repertoire is based on two human frameworks (one VH and one Vκ) that express well in bacteria and are frequently used in human antibodies. To achieve this, we first build dAb libraries, containing full synthetic diversity at key positions in the complementarity-determining regions (CDRs). We then use an antigen-independent preselection of this primary dAb repertoire on generic ligands of the VH and the Vκ scaffolds (namely, the bacterial superantigens, protein A and L) to enrich for folded dAbs. Finally, the CDRs of these preselected dAbs are randomly recombined to further expand genetic diversity. The resulting phage repertoire is in excess of 1010 clones and is largely populated by correctly folded (over 50%) functional dAbs.
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Acknowledgements
The authors were all employees of Domantis Ltd at the time this work was carried out. dAbTM and Domain AntibodyTM are all registered trademarks in the name of Domantis Limited.
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Ignatovich, O., Jespers, L., Tomlinson, I.M., de Wildt, R.M.T. (2012). Creation of the Large and Highly Functional Synthetic Repertoire of Human VH and Vκ Domain Antibodies. 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_4
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DOI: https://doi.org/10.1007/978-1-61779-968-6_4
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Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-61779-967-9
Online ISBN: 978-1-61779-968-6
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