Aims: Production of a monoclonal antibody (MAb) to Escherichia coli O157 to develop a rapid test using a sandwich ELISA (sELISA) format. Methods and Results: A MAb (7A6) was developed to the long-chain lipopolysaccharide of E. coli O157. A sELISA developed with the MAb reacted with 28 bovine and seven human enterohaemorrhagic E. coli (EHEC) O157 strains and also with two enterotoxigenic E. coli O157 strains. Cross-reaction to a rabbit diarrhoeal E.coli O15, Citrobacter freundii, Salmonella urbana and Vibrio cholerae O1 Inaba was detected. Conclusions: A MAb-based sELISA to detect E. coli O157 was produced. Its application to ®eld samples is required to fully determine its prospective use for the detection of EHEC O157, to evaluate the non-speci®c interference of the cross-reacting strains. Signi®cance and Impact of the Study: The assay produced is not wholly speci®c to EHEC O157, but has the potential to be used as a rapid method for screening large numbers of samples for E. coli O157.

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.

Aims: To compare a recently developed monoclonal antibody (MAb) based sandwich ELISA (sELISA) with an immunomagnetic separation (IMS) method for the detection of Escherichia coli O157 in bovine faeces. Methods and Results: Faecal samples from 345 cattle were obtained from eight farms in Northern Ireland, in which human disease due to E. coli O157 had occurred. Both assays detected E. coli O157 on ®ve of the farms and the phage-type of the majority of the bovine strains were the same as the corresponding human isolates. Similar numbers of the organism were detected by the two methods, 59 by the sELISA and 53 by the IMS procedure, 39 of the positive samples being common to both. Twenty samples were sELISA positive/IMS negative. Conclusions: If the IMS is regarded as the gold standard, then the sELISA is less sensitive and less speci®c, but under the conditions used sELISA positive results were obtained from all positive farms, and the sELISA gave a presumptive positive a day earlier than the IMS method. Signi®cance and Impact of the Study: The sELISA has the potential to be used as a rapid method for screening large numbers of samples for E. coli O157, but further work is required to determine its speci®city.

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