Generation and evaluation of nanobodies specific for virulence proteins from Vibrio parahaemolyticus causing translucent post-larvae vibriosis.

The bacterial pathogen Vibrio parahaemolyticus, specifically strains carrying the virulence genes vhvp-1, vhvp-2, and vhvp-3, is the causative agent of translucent post-larvae vibriosis (TPV) in shrimp. Afflicted shrimps display a distinct glass-like transparency, leading to the common moniker 'glass post-larvae'. This highly lethal disease triggers rapid onset mortality in aquaculture settings. Given the absence of effective therapeutic interventions, early detection remains the most critical strategy for disease management. To establish a rapid diagnostic strategy, our research focused on isolating and evaluating novel nanobodies that specifically recognize the primary TPV virulence proteins. We constructed and panned two distinct phage display libraries, one derived from alpacas and the other from sharks, to discover high-affinity binders. Following prokaryotic expression in an Escherichia coli system, the purified nanobodies underwent rigorous affinity and specificity profiling via western blot, enzyme linked immunosorbent assay (ELISA), and Bio-layer interferometry (BLI). We subsequently integrated the optimal nanobody candidates with colloidal gold immunochromatography to fabricate lateral flow test strips for on-site TPV monitoring. These engineered strips achieved visual detection limits of 12.5 μg/mL, 6.25 μg/mL, and 12.5 μg/mL for the VHVP-1, VHVP-2, and VHVP-3 antigens, respectively. Specificity evaluations revealed that the VHVP-2 and VHVP-3 strips possess high analytical specificity, while the VHVP-1 strip displayed a degree of cross-reactivity requiring further optimization. Nevertheless, by offering immediate, equipment-free diagnostic readouts, these colloidal gold test strips utilizing nanobodies provide a highly practical and scalable potential tool for rapid pathogen surveillance and disease management in the shrimp farming industry.