Nanobodies (Nbs) are the smallest known functional antibody fragments. Nbs are derived from the variable segment of camelid heavy chain‐only antibodies, known as VHH. Unlike conventional monoclonal antibodies, Nbs can recognize epitopes that are otherwise inaccessible, due to their small size(∼15 kDa) and unique structure. These properties make Nbs highly versatile, enabling their use in a wide range of applications, including therapeutics, diagnostics, and as molecular markers. Several display platforms, such as bacterial, phage, and yeast display, have been employed for Nb selection. Among these, Escherichia coli is particularly advantageous for cloning, amplification, and maintenance of large Nb repertoires due to its high transformation efficiency, stability of cloned DNA, and availability of numerous well-validated protein expression and secretion systems.
In this study, we report a technique for nanobody discovery by bacteria surface display library by leveraging flow cytometry to rapidly isolate nanobodies against various viral pathogens with a focus on flaviviruses and viral haemorrhagic fever viruses such as Japanese Encephalitis virus and Ebola virus. This system employs flow cytometric sorting of bacterial surface-expressed nanobody libraries cloned from immunized alpacas (Lama pacos L.). This allows high throughput and streamlined screening of a large range of library sizes. The resultant clonal selection is sequenced and cloned, engineered and produced in mammalian cells. The Nbs undergo screening for antigen-binding affinity and neutralization activity in vitro. This rapid pipeline for nanobody isolation against highly pathogenic viruses allows for rapid response of potential viral outbreaks through the development of therapeutics and diagnostics.