Development of Chicken Immunoglobulin Y for Rapid Detection of Cronobacter muytjensii in Infant Formula Powder

Abstract

The present study was aimed to produce a chicken polyclonal antibody against Cronobacter muytjensii and to develop an immunoassay for its detection. Purification of anti-C. muytjensii IgY from egg yolk was accomplished using various methods such as water dilution and salt precipitation. As a result, sodium dodecyl sulfate-polyacrylamide gel electrophoresis produced two bands around 30 and 66 kDa, corresponding to a light and a heavy chain, respectively. Indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) was performed to determine the effectiveness of the chicken IgY against C. muytjensii. The optimum conditions for detecting C. muytjensii by indirect ELISA and checkerboard titration of the antigen revealed an optimum average absorbance at the concentration of 18 µg/mL, having ca. 10 8 coated cells per well. The anti-C. muytjensii IgY antibody had high specificity for C. muytjensii and low cross-reactivity with other tested pathogens. In this assay, no cross-reactivity was observed with the other genera of pathogenic bacteria including Escherichia coli O157:H7, Salmonella Typhimurium, Staphylococcus aureus, Bacillus cereus, Enterobacter aerogenes, Salmonella Enteritidis and Listeria monocytogenes. In addition, detection of C. muytjensii in infant formula powder showed a low matrix effect on the detection curve of IC-ELISA for C. muytjensii, with similar detection limit of 10 5 CFU/mL as shown in standard curve. These findings demonstrate that the developed method is able to detect C. muytjensii in infant formula powder. Due to the stable antibody supply without sacrificing animals, this IgY can have wide applications for the rapid and accurate detection of C. muytjensii in dairy foods samples.

Development of an Indirect Non-Competitive Enzyme-Linked Immunosorbent Assay for the Detection of Cronobacter muytjensii in Infant Formula Powder

“…was not higher than 56% (Iversen et al 2008;Joseph et al 2012). On the other hand, Kim et al (2012) reported an indirect competitive enzyme linked immunosorbent assay by using a developed anti-chicken immunoglobulin Y (IgY) with a detection limit of 10 5 CFU/㎖ in pure culture. In terms of antibody availability, IgY could be a better option than IgG.…”