A robust nanobody-magnetic bead immunoassay for food safety monitoring: Sensitive detection of viral contamination in giant salamander.

The Chinese giant salamander is a valued food source of high-quality protein; however, the prevalence of Giant Salamander Iridovirus (GSIV) poses a significant threat to the safety and stability of the aquaculture food supply chain. Rapid and sensitive screening of viral pathogens in complex food matrices is therefore critical. We developed a bridge-free double-nanobody sandwich ELISA using magnetic beads (MBs) for detection of GSIV. AlphaFold 3 modeling suggested that covalent immobilization via conserved framework lysines may promote an optimal "upright" nanobody orientation, significantly enhancing capture efficiency compared with passive adsorption. The assay achieved a detection limit of 2.2 × 105 PFU/mL within 2 h, with sensitivity comparable to that of our previously reported pAb-based assay. The method exhibited exceptional resistance to matrix interference in undiluted tissue homogenates and was adapted to a semi-automated workflow. This work provides a scalable foundation for automated pathogen surveillance in aquatic food supply chains, ensuring food safety.