Study On A Sandwich Enzyme Linked Immunosorbent Assay For Detection Of Cronobacter Sakazakii

Cronobacter spp.known as an opportunistic pathogen have high fatality rate. It’s very meaningful for people healthy. Monoclonal antibodies (mAbs) were generated using 100℃ boiled ATCC29544 strain to immunize the animals. A sandwich ELISA using above-mentioned antibodies were developed for detection of Cronobacter sakazakii.The sandwich ELISA was performed as follows:0.5μg/well coating antibody 4℃ coated over night,0.5% milk 37℃ blocked 1.5 h,0.1% BSA-PBS diluted sample,1:2000 dilution detection antibody, second-antibody active 30 min.The LOD of the sandwich ELISA was 2×104 cfu/mL. Besides six species of Cronobacter spp. including Cronobacter sakazakii, Cronobacter malonaticus, Cronobacter turicensis, Cronobacter muytjensii, Cronobacter dublinensis and Cronobacter genomospeciesl and seven other Enterobacteriaceae including Escherichia coli, Proteus mirabilis, Citrobacter freundii, Enterobacter cloacae, Shigella flexneri, Enterobacter agglomerans and Salmonella enteritidis were tested in this assay. Accroding to the cross-reactivity experiment, the sandwich ELISA was specific to Cronobacter sakazakii.After 10 h enrichment, Cronobacter sakazakii could be detected as sensitive as 1.0 cfu/100g in infant formula based on the method. All the detection procedure can be performed in 3 h.It is demonstrated that the ELISA developed in this study are highly sensitive and rapid assay for screening powered infant formula for the presence of this highly virulent pathogenic Cronobacter sakazakii.