Channel-Like Binding Pocket-Mediated Nanobody-Alkaline Phosphatase Fusion Protein for Chemiluminescent Immunoassay of Anticoagulant Rodenticides.

Widespread application of anticoagulant rodenticides (ARs) has raised public health concerns due to their toxicity and persistence. Nanobody (Nb) fusion proteins integrate target recognition and signal output for facile immunoassay development. However, whether next-generation sequencing (NGS)-identified Nbs are equally suitable for fusion-protein construction and immunoassay development as phage display-derived Nbs remains underexplored because previous studies on Nb-fusion proteins have focused exclusively on phage display-derived Nbs. Here, two high-affinity anti-AR Nbs, phage display-derived AR-Nb and NGS-identified NGS-Nb, were compared using docking, simulation, and assessments of Nb-fusion-protein binding performance. AR-Nb formed a more stable DIF complex via a channel-like pocket and localized interactions, and AR-Nb-ALP produced ∼5-fold stronger signals than NGS-Nb-ALP. The optimized AR-Nb-ALP-based chemiluminescent enzyme immunoassay (CLEIA) detected four ARs in urine, soil, and river water with LODs of 0.26-1.03 ng/mL and recoveries of 70.3-99.9%. This work provides a novel perspective on how different selection routes affect nanobody structure and fusion-protein performance.