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.
CITATION STYLE
Lowden, M. J., van Faassen, H., Raphael, S., Ryan, S., Hussack, G., & Henry, K. A. (2022). Facile Affinity Maturation of Single-Domain Antibodies Using Next-Generation DNA Sequencing. In Methods in Molecular Biology (Vol. 2446, pp. 245–268). Humana Press Inc. https://doi.org/10.1007/978-1-0716-2075-5_12
Mendeley helps you to discover research relevant for your work.