Preparación, caracterización y mecanismo de unión de nanobodies dirigidos a múltiples serotipos de Salmonella

Salmonella Enteritidis and Salmonella Typhimurium both are the most prevalent Salmonella serotypes responsible for human infections. Immunoassays, typically relying on serotype-specific antibodies, often lack the capacity for broad-spectrum Salmonella serotype detection. To address this limitation, in this study, nanobody VHH-II-3 was isolated from a phage display library constructed by immunization with inactivated S. Enteritidis and S. Typhimurium cells, followed by a cross-antigen strategy for biopanning. The VHH-II-3 nanobody could bind against 13 Salmonella serotypes and exhibited excellent thermostability. The antigenic targets of VHH-II-3 were identified as the structural epitopes of flagellar proteins FliC and FlgL through His-tag pull-down assays coupled with mass spectrometry, further verified by Western blotting and VHH ELISA. Biolayer interferometry (BLI) affinity analysis revealed that the binding constant of VHH-II-3 to FliC reached the nanomolar level (10^-9 M), with the amino acid residue of Gln105 within the CDR3 loop playing a critical role in antigen recognition. Notably, FlgL likely contributed significantly to the broad-spectrum recognition capability of VHH-II-3, as it was highly conserved across diverse Salmonella serotypes. This study provided a promising nanobody for the broad-serotype detection for Salmonella and offered novel perspectives into their antigen-binding mechanisms for improved diagnostic and monitoring applications.