Development of a Sandwich ELISA for EHEC O157:H7 Intimin γ1.
Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a zoonotic pathogen of worldwide importance that causes foodborne infections in humans. Intimin gamma 1 (intimin γ1) is one of the most important outer membrane proteins required for EHEC's intimate adhesion to epithelial cells. Here, we generated a polyclonal antibody (pAb) and a monoclonal antibody (mAb) against intimin γ1 to develop a double antibody sandwich ELISA (DAS-ELISA) with increased sensitivity and specificity for measuring EHEC O157:H7. To achieve this goal, a rabbit pAb was used as a capture antibody, and a mouse mAb was a detection antibody. No cross-reactivity was observed with the other genera of pathogenic bacteria tested with the DAS-ELISA, which included Salmonella enteritidis, Shigella flexneri type 2, Listeria monocytogenes, Streptococcus suis type 2, and other 18 serotype E. coli. Detection limits of the DAS-ELISA were 1 × 103 CFU/mL for EHEC O157:H7 cultures, 1 × 104 CFU/g before enrichment, and 1 × 102 CFU/g after enrichment of contaminated samples. Field samples (n = 498) were tested using a previously established duplex-PCR method and compared to our DAS-ELISA. The DAS-ELISA had a specificity of 94.4%, a sensitivity of 91.5% and accuracy of 94.0% compared with duplex-PCR. The DAS-ELISA developed here can be applied to EHEC O157:H7 quantification in food, animal, and environmental samples.
Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Key Laboratory of Engineering Research of Veterinary Bio-products of Agricultural Ministry, National Center for Engineering Research of Veterinary Bio-products, Nanjing 210014, China, Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, 225009, China, Key Lab of Food Quality and Safety of Jiangsu Province-State Key Laboratory Breeding Base, Nanjing 210014, China.
Xuehan
http://orcid.org/0000-0002-0647-2266
X
Zhang
Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Key Laboratory of Engineering Research of Veterinary Bio-products of Agricultural Ministry, National Center for Engineering Research of Veterinary Bio-products, Nanjing 210014, China, Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, 225009, China, Key Lab of Food Quality and Safety of Jiangsu Province-State Key Laboratory Breeding Base, Nanjing 210014, China.
Meng
M
Li
Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Key Laboratory of Engineering Research of Veterinary Bio-products of Agricultural Ministry, National Center for Engineering Research of Veterinary Bio-products, Nanjing 210014, China, Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, 225009, China, Key Lab of Food Quality and Safety of Jiangsu Province-State Key Laboratory Breeding Base, Nanjing 210014, China.
Bicheng
B
Zhang
Department of Diagnostic Medicine/Pathobiology, Kansas State University, Manhattan, KS 66506, United States of America.
Kangming
K
Chen
Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Key Laboratory of Engineering Research of Veterinary Bio-products of Agricultural Ministry, National Center for Engineering Research of Veterinary Bio-products, Nanjing 210014, China, Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, 225009, China, Key Lab of Food Quality and Safety of Jiangsu Province-State Key Laboratory Breeding Base, Nanjing 210014, China.
Kongwang
K
He
D018829:Adhesins, Bacterial / Q000276:immunology* / Q000302:isolation & purification; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001201:Ascites / Q000378:metabolism; D003429:Cross Reactions / Q000276:immunology; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D019453:Escherichia coli O157 / Q000276:immunology*; D029968:Escherichia coli Proteins / Q000276:immunology* / Q000302:isolation & purification; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity
D018829:Adhesins, Bacterial; D000911:Antibodies, Monoclonal; D029968:Escherichia coli Proteins; C073819:eaeA protein, E coli