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Background 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 methods. The detection methods based on nucleic acid and conventional antibodies are fraught with limitations. Many of these limitations of the conventional antibodies can be circumvented using natural nanobodies which are endowed with characteristics, such as high affinity, thermal stability, easy production, especially higher diversity. This study aimed to select the special nanobodies against  S. Enteriditidis  for developing an improved nanobody-horseradish peroxidase-based sandwich ELISA to detect  S. Enteritidis  in the practical sample. The nanobody-horseradish peroxidase fusions can help in eliminating the use of secondary antibodies labeled with horseradish peroxidase, which can reduce the time of the experiment. Moreover, the novel sandwich ELISA developed in this study can be used to detect  S. Enteriditidis  specifically and rapidly with improved sensitivity. Results 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 × 10 4  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. Graphic Abstract

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