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

Xuehan Zhang,Meng Li,Kongwang He,Bicheng Zhang,Kangming Chen,George-John Nychas

doi:10.1371/journal.pone.0162274

Abstract

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.

Highlights

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a zoonotic pathogen of worldwide importance that causes foodborne infections in humans [1]
EHEC O157:H7 is characterized by the Intimin γ1 subtype and its C-terminal fragment is used as a target to detect EHEC O157:H7
SDS-PAGE showed that C1-intimin γ1 (32.03 kDa) and C2-intimin γ1 (13.60 kDa) were successfully expressed with 20 and 35% proportion to whole bacterial protein in contrast to naïve bacteria

Summary

Introduction

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a zoonotic pathogen of worldwide importance that causes foodborne infections in humans [1]. Ruminants, which are asymptomatic carriers, are considered to be a major source of EHEC O157:H7, which is spread through fecal contamination of food (i.e. animal meat, milk, and vegetables) [2]. Intimin is encoded by the E. coli attaching and effacing (eae) gene, which is required for intimate adhesion to epithelial cells and cytoskeletal reorganization. The N-terminal region of the Intimin molecule is conserved, and the variable C-terminal sequence of Intimin defines at least. 27 different Intimin subtypes [3,4,5]. EHEC O157:H7 is characterized by the Intimin γ1 subtype and its C-terminal fragment is used as a target to detect EHEC O157:H7

Methods

Results

Conclusion