Identification of novel monoclonal antibodies targeting the outer membrane protein C and lipopolysaccharides for Escherichia coli O157:H7 detection

Abstract

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.

Comparative immunogenicity of outer membrane protein K and whole‐cell antigens of Vibrio parahaemolyticus for diagnosis

“…Specifically, there were more polysaccharides bands (core structures and lipid A) at 15–20 kDa (Wang et al . 2021) in Western blot (WB).…”

Identification of a specific surface epitope of OmpC for Escherichia coli O157:H7 with protein topology facilitated affinity mass spectrometry

“…To better understand the specificity of mAb 2G12 for ECO157, we further investigated the sequence homology of the three extracellular loops with other tested non-O157 strains that are available in NCBI. These non-O157 strains did not react with mAb 2G12 at 6 log 10 CFU/mL in our previous study (Wang et al 2021 ). Results in Fig.…”

“…The direct identification of the target protein band (~ 35 kDa) of ATCC 35,150 in the SDS-PAGE gel by in-gel digestion and the LC–MS/MS also showed that the leading protein was OmpC (Supplemental Table S3 ). As reported previously by Wang et al ( 2021 ), the 70 kDa band was only observed for ECO157 CICC 21,530 and the ~ 35 kDa band was mainly from other ECO157 strains. All of these above results revealed that OmpC was the target outer membrane protein of mAb 2G12.…”

“…Between each WB step, membranes were washed four times in 15 mL of 10 mM phosphate-buffered saline with tween 20 (PBST) for 3 min of gentle shaking. Fifteen microliters of the antibody 2G12 (0.3 μg/mL) in PBST was added and incubated with the PVDF membrane at 25 ℃ for 1 h. The preparation and detailed message of mAb 2G12 was described in our previous work (Wang et al 2021 ). Fifteen microliters of the goat anti-mouse IgG antibody (0.2 μg/mL) in PBST was added and allowed to react at 25 ℃ for 1 h. After four times of washing as described above, 10 mL of the commercial TMB substrate for Western blot (GBCBIO, Guangzhou, China) was added and allowed to react for 5 min at 25 ℃.…”

“…The tested non-ECOO157 strains, which did not have cross-reactions with mAb 2G12, included E. coli O121, O111, O103, O45, O26, O101, O86, O78, O60, O25, O9, O6, O2, Shigella Sp., Citrobacter amalonaticus , Enterobacter cloacae , Campylobacter Sp, Aeromonas Sp., Vibrio Sp., Proteus Sp., Listeria monocytogenes , Staphylococcus aureus , and Bacillus subtilis . Initial evaluations indicated that the target of this mAb could be an outer member protein (Omp) with a size of approximately 35 kDa (Wang et al 2021 ).…”