Development of nanobody-horseradish peroxidase-based sandwich ELISA to detect Salmonella Enteritidis in milk and in vivo colonization in chicken

Salmonella Enteritidis (S. Enteritidis) being one of the most prevalent foodborne pathogens worldwide poses a serious threat to public safety. Prevention of zoonotic infectious disease and controlling the risk of transmission of S. Enteriditidis critically requires the evolution of rapid and sensitive detection

This newly developed a sandwich ELISA that used the SE-Nb9 as capture antibody and horseradish peroxidase-Nb1 to detect S. Enteriditidis in the spike milk sample and to analyze the colonization distribution of S. Enteriditidis in the infected chicken. These results demonstrated that the developed assay is to be applicable for detecting S. Enteriditidis in the spiked milk in the rapid, specific, and sensitive way. Meanwhile, the developed assay can analyze the colonization distribution of S. Enteriditidis in the challenged chicken to indicate it as a promising tool for monitoring S. Enteriditidis in poultry products. Importantly, the SE-Nb1-vHRP as detection antibody can directly bind S. Enteritidis captured by SE-Nb9, reducing the use of commercial secondary antibodies and shortening the detection time. In short, the developed sandwich ELISA ushers great prospects for monitoring S. Enteritidis in food safety control and further commercial production.

This study screened four nanobodies from an immunized nanobody library, after four rounds of screening, using the phage display technology. Subsequently, the screened nanobodies were successfully expressed with the prokaryotic and eukaryotic expression systems, respectively. A sandwich ELISA employing the SE-Nb9 and horseradish peroxidase-Nb1 pair to capture and to detect S. Enteritidis, respectively, was developed and found to possess a detection limit of 5 × 104 colony forming units (CFU)/mL. In the established immunoassay, the 8 h-enrichment enabled the detection of up to approximately 10 CFU/mL of S. Enteriditidis in milk samples. Furthermore, we investigated the colonization distribution of S. Enteriditidis in infected chicken using the established assay, showing that the S. Enteriditidis could subsist in almost all parts of the intestinal tract. These results were in agreement with the results obtained from the real-time PCR and plate culture. The liver was specifically identified to be colonized with quite a several S. Enteriditidis, indicating the risk of S. Enteriditidis infection outside of intestinal tract. Conclusions: This newly developed a sandwich ELISA that used the SE-Nb9 as capture antibody and horseradish peroxidase-Nb1 to detect S. Enteriditidis in the spike milk sample and to analyze the colonization distribution of S. Enteriditidis in the infected chicken. These results demonstrated that the developed assay is to be applicable for detecting S. Enteriditidis in the spiked milk in the rapid, specific, and sensitive way. Meanwhile, the developed assay can analyze the colonization distribution of S. Enteriditidis in the challenged chicken to indicate it as a promising tool for monitoring S. Enteriditidis in poultry products. Importantly, the SE-Nb1-vHRP as detection antibody can directly bind S. Enteritidis captured by SE-Nb9, reducing the use of commercial secondary antibodies and shortening the detection time. In short, the developed sandwich ELISA ushers great prospects for monitoring S. Enteritidis in food safety control and further commercial production.