Abstract

Listeriolysin O (LLO), a crucial virulence factor of Listeria monocytogenes , endangers human health through contaminated refrigerated foods. In this study, we developed high-affinity monoclonal antibodies (mAbs) for sensitive LLO detection in foods, alongside characterizing antibody-antigen interactions. Soluble LLO was expressed in Escherichia coli and used as the antigen. The mAb pair (7D3–3H5) was selected from 12 candidates via mouse immunization and hybridoma screening. The genes encoding the variable heavy (VH) and light (VL) chains of 3H5 and 7D3 were cloned; subsequent molecular docking identified critical binding residues (3H5: GLN3, ASN104, SER49; 7D3: TYR32, TYR57), offering insights for antibody optimization. An antibody-based gold immunochromatographic strip (GICS) was established for rapid on-site detection, with visual limits of detection (vLOD) of 5 ng/mL (milk), 1 ng/g (vegetables), and 10 ng/g (pork). This work promotes the understanding of antibody-LLO interactions for future engineering and provides an effective tool for monitoring LLO contamination.