Highly sensitive ELISA for determination of nifursol metabolite in food samples without derivatization: From rational hapten design to molecular recognition mechanism.

High-affinity antibodies to nitrofuran metabolites could avoid derivatization, but existing nitrofuran haptens yield low-affinity antibodies due to poor immunogenicity. Herein, we selected 3,5-dinitrosalicylic acid hydrazide (DNSH), the metabolite of nifursol, as model analytes to produce high-affinity antibodies against metabolites. Three novel DNSH haptens were rationally designed, with aromatic haptens specially incorporating nitro group to enhance immunogenicity. After rationality evaluation by computational chemistry, antisera results indicated that nitro-modified haptens, DNSH-1 and DNSH-2, induced a stronger immune response. Furthermore, high-affinity mAb 2H6 was obtained from DNSH-1 with IC of 0.16 ng mL for DNSH. Molecular simulations demonstrated that abundant electrostatic characteristics and hydrogen bonds of nitro group contributed to its immune-enhancing effect. Finally, a rapid and sensitive icELISA was developed with limit of detections of 0.1-0.5 μg kg in pork, chicken and fish meat. The total assay time was less than 3 h, eliminating prolonged 16-h derivatization and significantly improves detection efficiency.