Development of monoclonal antibody and immunoassays for determination of florylpicoxamid in foods: A sub-similarity principle for guiding the hapten design

Florylpicoxamid (FLCD) is a novel broad-spectrum fungicide with considerable application potential. However, its residues in food may pose health risks, and a rapid detection method is currently lacking. Although immunoassays are well-established for detecting small-molecule contaminants, the development of corresponding antibodies for FLCD has been hampered by the complex and cyanide-dependent synthesis of its traditional haptens. To overcome this limitation, this study introduces a sub-similarity principle for rational hapten design. Guided by computational chemistry, this approach involves designing haptens that retain the feature structure essential for eliciting specific immune responses, while substituting the distal molecular regions with simpler, lower-cost, and more synthetically accessible analogues. Subsequently, three FLCD haptens featuring distinct spacer arms were designed, and the first monoclonal antibody (mAb) specific to FLCD was successfully developed, with an IC₅₀ of 0.6 μg/L and negligible cross-reactivities below 0.1 % to other 5 picolinamide pesticides. Investigation of the mAb's recognition mechanism further confirmed the reliability of the sub-similarity principle. Utilizing this mAb, an indirect competitive enzyme-linked immunosorbent assay was developed for the rapid screening of FLCD residues in tomatoes, grapes, milk, and chicken, achieving limits of detection (LODs) ranging from 0.52 to 1.46 μg/L. Furthermore, a lateral flow immunoassay was constructed for on-site detection, yielding visual LODs of 1.0-3.0 μg/L and cut-off values of 1.2-4.0 μg/L. This study not only presents a novel strategy for generating antibodies against small molecules, but also addresses a critical gap in the rapid detection of FLCD.