Detection of Listeria monocytogenes using anti-internalin antibodies generated through epitope prediction

Listeria monocytogenes is a major food borne pathogen, and rapid, highly specific immunodetection tools are essential for surveillance in dairy food systems. This study reports an epitope-guided strategy for developing anti-Internalin A (Inl A) and B (Inl B) antibodies and their application in ELISA based detection of of L. monocytogenes. The inlA gene was successfully amplified from three reference strains, confirming target sequence conservation within outbreak associated serotypes. The 19 amino acid, highly hydrophilic and antigenic peptides from the surface exposed region of the Inl A and 21 amino acids with same property of Inl B proteins were identified through multi-parameter in silico prediction and used to immunize the rabbits. The resulting polyclonal antibodies exhibited strong peptide specific binding, with significantly increased titer across successive bleeds (p < 0.05). Purified Inl A/ Inl B antibodies demonstrated selective recognition of L. monocytogenes in dot blot / ELISA formats, with limited cross reactivity primarily attributable to Staphylococcus aureus Protein A interference. Indirect ELISA showed strong linear correlation between optical density and bacterial concentration (R² = 0.982), with a detection limit of 4.30 ± 0.02 CFU/ml. A dual epitope sandwich ELISA employing anti-Inl A (capture) and anti-Inl B (Detection) further improved specificity and achieved a detection limit of 4 log CFU/ml, while effectively distinguishing L. monocytogenes from closely related L. ivanovii and non-pathogenic L. innocua. When applied to artificially contaminated milk following short enrichment, sandwich ELISA detected inocula as low as 1.15 log cfu/ml, with matrix independent assay performance. This work demonstrates, for the first time, an epitope predicted Inl A/ Inl B antibody that reliably discriminates pathogenic Listeria species and supports sensitive detection in dairy matrices. The combined Inl A/ Inl B targeting strategy offers a robust, translationally relevant platform for foodborne pathogens screening and routine milk safety monitoring.