Abstract: Purpose: To develop a rapid detection kit for Enterobacter sakazakii contamination in milk powder. Methods: A double-antibody sandwich ELISA assay was developed using two monoclonal antibodies against E. sakazakii as the capture antibody and detection antibody, respectively. A test kit that could show a color reaction within 2 h was developed through the optimization of the ELISA reaction. Results: The optimum working concentration of the capture antibody was 10 μg/mL and the optimum dilution of the detection antibody was 1:5 000. The limit of detection (LOD) of the kit was 104 CFU/mL and a linear relationship between OD450 nm values and the lg values of E. sakazakii concentration was observed in the range of 104–108 CFU/mL. In specificity test, three reference strains of E. sakazakii could be detected by the kit whereas other nine strains of common foodborne pathogens were detected as negative. The LOD of the kit was substantially improved to 1 CFU/mL of the original bacterial content after pre-incubation of model samples in broth for 8 h. Both the intra-assay and inter-assay coefficients of variation of the kit were lower than 6%. It could be stored at room temperature for at least six months. Conclusions: The established double-antibody sandwich ELISA kit is suitable for the rapid detection of E. sakazakii in milk powder with high sensitivity, high specificity, high precision and strong stability.

Key words: double-antibody sandwich, ELISA kit, sensitive, rapid assay, pathogenic bacteria