Screening and characterization of anti-Listeria monocytogenes inlG-protein nanobodies and development of an ELISA method

Abstract

Listeria monocytogenes (LM) is one of the eight species within the Listeria genus and one of the most lethal foodborne pathogens. Accurate and rapid detection is crucial for preventing infections caused by this bacterium. Nanobodies, with the advantages of strong affinity, high specificity, low off-target effects, excellent thermal stability, and good solubility, have emerged as a novel reagent for pathogen detection. InlG (Internalin G), a surface-anchored virulence protein accessible on the bacterial surface, was selected as the target antigen to ensure high specificity and effective antibody binding. This study expressed and purified the inlG protein by prokaryotic expression. High-specificity nanobodies targeting the inlG protein were screened using phage display technology and were then used as capture antibodies to establish an indirect sandwich ELISA detection method. The established indirect sandwich ELISA assay demonstrates excellent reproducibility (intra-assay CV: 2.39% and inter-assay CV: 2.78%) and high specificity, with slight cross-reaction with Escherichia coli, and no significant cross-reaction with other food-borne pathogens. The limit of detection (LOD) was 1.0 × 10⁵ CFU mL⁻¹ and limit of quantitation (LOQ) was 3.0 × 10⁵ CFU mL⁻¹ (S/N = 10, CV ≤ 10%). Spiked recovery rates ranged from 96.0% to 107.5% in five food matrices (beef, fish, milk, soft cheese, and deli turkey), with an RSD ≤ 4.68%. Furthermore, these nanobodies targeting InlG may serve as penicillin for controlling L. monocytogenes infections, and chemical residues benzylpenicilloic acid (BPNLA) risk, addressing the challenge of antibiotic resistance.