Computational Hapten Rationalization Unlocks Multiplex Lateral Flow Immunoassay for Quinolines

Computation-aided spacer-mismatch epitope engineering is implemented to generate highly specific monoclonal antibodies targeting two hazardous quinoline pollutants, hydroxyquinoline (HQL) and nitroxoline (NXO). These structurally similar quinoline derivatives are widely misused in agricultural and animal husbandry, bringing severe hidden risks to food and ecological environments. Molecular descriptors reflecting epitope exposure and skeleton integrity were adopted to screen optimal alkyl spacer lengths for rational hapten design. Combined with the hybridoma technology, two monoclonal antibodies with high affinity and specificity were successfully obtained. Half-maximal inhibitory concentrations determined by the indirect competitive enzyme-linked immunosorbent assay were 1.104 ng/mL and 1.671 ng/mL for HQL and NXO, respectively. Furthermore, a dual-color latex microsphere-based multiplex lateral flow immunoassay was developed for simultaneous detection of HQL and NXO in apple, chicken, and livestock wastewater. The proposed method achieved matrix-matched limits of detection of 5.898 μg/kg and 6.289 μg/kg in apple, 6.305 μg/kg and 7.279 μg/kg in chicken, and 2.450 μg/kg and 3.879 μg/kg in livestock wastewater for HQL and NXO, respectively. The method was validated via real sample analysis, demonstrating favorable accuracy and reliability, which was suitable for rapid and high-throughput on-site screening.