Use of a Monoclonal Antibody against anEscherichia coliO26 Surface Protein for Detection of Enteropathogenic and Enterohemorrhagic Strains

Abstract

A monoclonal antibody (MAb) was obtained from a mouse immunized with solubilized outer membrane proteins extracted from a bovine enterohemorrhagic strain of Escherichia coli (EHEC), O26. The MAb produced a strong immunoblot reaction at approximately 21 kDa for an O26 strain containing the intimin gene (eae) and verocytotoxin (VT), but not with an O26 eae- and VT-negative strain, or O157 eae- and VT-positive strains. The MAb was used in a sandwich enzyme-linked immunosorbent assay (ELISA) format to screen strains from animal and human sources, and all reactive strains were characterized for the presence of eaeand the gene encoding VT factors by PCR. The antigen was detected in a group of strains containing a high proportion of O26, the majority of which were eae positive with or without VT; these were isolated mostly from animal enteritis cases but included a small number of human enteric isolates. Nonreactors includedeae-positive (with or without VT) O157 strains and one O26 strain. In a survey of mixed cultures from both animal and human enteric disease, ELISA-positive reactions were obtained from 7.1 to 11.2% of samples from bovine, porcine, ovine, and human sources. The two human O8 and ten animal O26 ELISA-reactive pure strains obtained from these samples contained six eae- and/or VT-positive strains; the other six strains lost their ELISA positivity following storage at −70°C, after which none were found to contain eithereae or VT factors. The association of the antigen detected by the MAb with significant enteropathogenic E. coli and EHEC virulence factors in isolates from both animal and human enteric infections indicates a diagnostic potential for the assay developed.

Development of a monoclonal sandwich ELISA for the detection of animal and human Escherichia coli O157 strains

“…This MAb, 7A6, was selected for further investigation in a sELISA format, as described previously (Ball et al . 1993; Kerr et al . 1999).…”

Development of a capture/enrichment sandwich ELISA for the rapid detection of enteropathogenic and enterohaemorrhagic Escherichia coli O26 strains

“…The wells were washed, broth culture medium was added and the plates were incubated at 37°C overnight to allow for the multiplication of captured cells. This capture/enrichment sELISA was developed using an E. coli O26‐specific MAb, 2F3 (Kerr et al. 1999), and an E. coli O26‐specific polyclonal rabbit serum.…”

“…Therefore to identify the reservoirs of EHEC O26 strains and the routes of transmission to humans, sensitive detection methods are required (De Boer and Heuvelink 2000; Bettelheim and Beutin 2003). The present study describes the development of a sandwich ELISA (sELISA) for the detection of E. coli O26, using a monoclonal antibody (MAb) that has been shown to be largely EPEC/EHEC specific (Kerr et al. 1999), and using selective enrichment as a means to improve the sensitivity of the assay.…”

Variability in Characterizing Escherichia coli from Cattle Feces: A Cautionary Tale

“…Discrepancies between molecular methods and TS have been previously reported [ 26 ] as has cross-reactivity in antibodies used in TS [ 14 , 27 , 28 ]. However, heightened discrepancies between TS and PCR for O26 and O45 are puzzling when compared to other serogroups.…”