Facile Affinity Maturation of Single-Domain Antibodies Using Next-Generation DNA Sequencing.

Autor:	Lowden MJ; Human Health Therapeutics Research Centre, Life Sciences Division, National Research Council Canada, Ottawa, ON, Canada., van Faassen H; Human Health Therapeutics Research Centre, Life Sciences Division, National Research Council Canada, Ottawa, ON, Canada., Raphael S; Human Health Therapeutics Research Centre, Life Sciences Division, National Research Council Canada, Ottawa, ON, Canada., Ryan S; Human Health Therapeutics Research Centre, Life Sciences Division, National Research Council Canada, Ottawa, ON, Canada., Hussack G; Human Health Therapeutics Research Centre, Life Sciences Division, National Research Council Canada, Ottawa, ON, Canada., Henry KA; Human Health Therapeutics Research Centre, Life Sciences Division, National Research Council Canada, Ottawa, ON, Canada. kevin.henry@nrc-cnrc.gc.ca.; Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, ON, Canada. kevin.henry@nrc-cnrc.gc.ca.
Jazyk:	angličtina
Zdroj:	Methods in molecular biology (Clifton, N.J.) [Methods Mol Biol] 2022; Vol. 2446, pp. 245-268.
DOI:	10.1007/978-1-0716-2075-5_12
Abstrakt:	Binding affinity is one of the primary determinants of antibody function. Here, we provide a protocol for simple and rapid affinity maturation of single-domain antibodies (sdAbs) using tandem phage display selection and next-generation DNA sequencing. The sequence of a model camelid sdAb directed against Clostridioides difficile toxin A (A26.8) was diversified using either random or site-saturation mutagenesis and cloned into a phagemid vector upstream of gene 3. The resulting phage-displayed sdAb libraries were panned against C. difficile toxin A and the panning outputs interrogated using Illumina MiSeq sequencing. Through bioinformatic analyses, we were able to identify individual affinity-enhancing amino acid substitutions in the sdAb complementarity-determining regions that, when combined, resulted in affinity improvements of approximately 10-fold. The advantages of this method are that it does not require extensive screening and characterization of individual clones, nor structural information on the mechanism of the sdAb:antigen interaction. (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)
Databáze:	MEDLINE
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