Preparation, Characterization, and Binding Mechanism of Nanobodies Targeting Multiple Serotypes of 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.