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

Methods Mol Biol. 2022:2446:245-268. doi: 10.1007/978-1-0716-2075-5_12.

Abstract

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.

Keywords: Affinity maturation; Antibody; Next-generation DNA sequencing; Phage display; Single-domain antibody; VHH.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antibody Affinity
  • Cell Surface Display Techniques / methods
  • Clostridioides difficile* / genetics
  • High-Throughput Nucleotide Sequencing / methods
  • Peptide Library
  • Sequence Analysis, DNA
  • Single-Domain Antibodies* / chemistry

Substances

  • Peptide Library
  • Single-Domain Antibodies