Abstract
Background and Aim: Listeria monocytogenes remains a major foodborne pathogen globally, with mortality rates ranging from 20%–40%. The increasing incidence of listeriosis and the limitations of culture-based and polymerase chain reaction-based diagnostics highlight the need for rapid, cost-effective, and highly specific immunoassays. This study aimed to develop and validate a regionally adapted sandwich enzyme-linked immunosorbent assay (ELISA) based on monoclonal and polyclonal antibodies (pAbs) raised against a recombinant p60 antigen derived from a Kazakhstani L. monocytogenes field isolate. Materials and Methods: The p60 gene lacking its N-terminal signal peptide was amplified from a regional L. monocytogenes isolate, cloned into the pET28c(+) vector, and expressed in Escherichia coli arctic express (DE3). Recombinant p60 protein was purified by Ni2⁺-affinity chromatography and used to immunize BALB/c mice and Chinchilla rabbits for monoclonal antibodies and pAbs antibody production. Hybridoma clones were screened for specificity using indirect ELISA and Western blot. A sandwich ELISA was assembled using mAb 1H8 as the capture antibody and horseradish peroxidase-conjugated rabbit pAbs as detection antibodies. Analytical sensitivity and diagnostic performance were evaluated using serial dilutions of recombinant p60 and culture supernatants of L. monocytogenes isolates recovered from 507 food samples. Results: The recombinant p60 antigen (50.3 kDa) was successfully expressed and purified at 5.9 mg/L yield. Among seven stable hybridoma clones, mAb 1H8 exhibited the highest affinity (Ka = 2.5 × 1010 M⁻1) and specificity without cross-reactivity to non-Listeria bacteria. The optimized sandwich ELISA achieved a detection limit of 1.5 ng/mL, corresponding to approximately 103 colony-forming units/mL. All six L. monocytogenes field isolates tested positive in the assay, with results strongly correlating with viable cell counts (R2 = 0.89). The assay demonstrated comparable sensitivity to commercial kits while offering shorter assay time (2 h) and substantially lower production cost. Conclusion: The developed sandwich ELISA provides a sensitive, specific, rapid, and regionally tailored diagnostic platform for detecting pathogenic L. monocytogenes in food samples. By integrating locally produced recombinant antigens and immunoreagents, the assay offers a cost-effective alternative to imported kits, supporting national food safety programs and One Health surveillance initiatives. Keywords: food safety surveillance, foodborne pathogens, hybridoma technology, immunodiagnostics, Listeria monocytogenes, monoclonal antibodies, One Health, p60 antigen, polyclonal antibodies, recombinant protein, sandwich enzyme-linked immunosorbent assay.