Aims To identify and evaluate the application of two novel monoclonal antibody (mAb) 2G12 against outer membrane protein (Omp) C and mAb 12B1 targeting the O chain of the lipopolysaccharides (LPS) of Escherichia coli O157:H7 (ECO157). Methods and Results The sensitivity and specificity of these two antibodies were evaluated with eight ECO157 strains and 68 untargeted strains. mAb 2G12 and 12B1 had no detectable binding with any of the non‐O157 strains at 6·0 log10 CFU per ml, while its high specificity and affinity remained with all ECO157 strains. When a higher level (8·0 log10 CFU per ml) was tested, 2G12 and 12B1 did not react with 82·35 and 97·06% of the non‐O157 strains respectively. Based on the pair of two antibodies, the sandwich enzyme‐linked immunosorbent assay detected 100% (8/8) of ECO157 strains and none of the non‐ECO157 strains. The detection limit of ECO157 strains in pure culture were 4·2 ± 0·2 log10 CFU per ml. When the developed test was applied to artificially inoculated beef samples, the detection limit was 6·0 log10 CFU per gram without enrichment and 1·0 log10 CFU per gram after 12 h of enrichment. Conclusions The two novel antibodies identified in this study served as great candidates for the recovery, and detection of ECO157 from different environmental and food samples. Significance and Impact of the Study ECO157‐specific detection was improved by a combination of the novel OmpC mAb and LPS mAb with defined target antigen and good specificity.

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