Development of a Sandwich ELISA for EHEC O157:H7 Intimin γ1

作者：Zhang, Xuehan^*; Li, Meng; Zhang, Bicheng; Chen, Kangming; He, Kongwang^*

来源：PLos One, 2016, 11(9): e0162274.

DOI：10.1371/journal.pone.0162274

• ESCHERICHIA-COLI O157H7
• REAL-TIME PCR
• EPEC
• IDENTIFICATION
• ANTIBODIES
• O157-H7
• GENES
• CELLS

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a zoonotic pathogen of worldwide importance that causes foodborne infections in humans. Intimin gamma 1 (intimin gamma 1) 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 gamma 1 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 x 10(3) CFU/mL for EHEC O157:H7 cultures, 1 x 10(4) CFU/g before enrichment, and 1 x 10(2) 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.

• 出版日期2016-9-7
• 单位江苏省农业科学院