| Abstract [eng] |
Nanobodies, also known as VHH domains, are the smallest antigen-binding fragments, derived from camelid heavy-chain-only antibodies (HcAbs). Due to their compact structure, stability, and high specificity, they are increasingly recognized as versatile affinity ligands in biotechnology. However, depending on the intended application, their binding properties may require further improvement, creating a need for targeted optimization strategies. The aim of this study was to develop an experimental framework for site-directed mutagenesis- based nanobody affinity optimization, enabling the functional evaluation of selected protein regions involved in antigen recognition. The work included bioinformatic sequence analysis, site-directed mutagenesis of selected amino acid positions, recombinant expression of mutant nanobody variants, and assessment of their interaction with the target molecule. This approach provides a framework for rational nanobody engineering when experimentally determined structural information is not available. Overall, the work contributes to the development of systematic strategies for nanobody optimization in biotechnological applications. |
