Production and characterization of monoclonal antibodies specific for the lipopolysaccharide of Escherichia coli O157

Abstract

Identification of the O157 antigen is an essential part of the detection of Escherichia coli O157:H7, which is recognized as a major etiologic agent of hemorrhagic colitis. However, polyclonal antibodies produced against E. coli O157:H7 lipopolysaccharide (LPS) may react with several other bacteria including Brucella abortus, Brucella melitensis, Yersinia enterocolitica O9, Escherichia hermannii, and Stenotrophomonas maltophilia. We produced eight monoclonal antibodies (MAbs) specific for the LPS of E. coli O157. Western blots (immunoblots) of both the phenol phase (smooth) and the aqueous phase (rough) of hot phenol-water-purified LPS indicated that three of the MAbs were specific for the O antigen and five were reactive with the LPS core. The eight MAbs could be further differentiated by their reactivities to Salmonella O30 LPS (group N), which is reported to be identical to the E. coli O157 antigen. All eight MAbs reacted strongly to all of the 64 strains of E. coli O157 tested, which included 47 isolates of O157:H7 and 17 other O157 strains. None of the eight MAbs cross-reacted with any of the 38 other E. coli serotypes tested, which consisted of 29 different O-antigen serotypes, or with 38 strains (22 genera) of nonE. coli gram-negative enteric bacteria. Escherichia coli O157:H7 has been identified as a major etiologic agent of hemorrhagic colitis (31), which is occasionally accompanied by serious complications such as hemolyticuremic syndrome (18, 24) or thrombic thrombocytopenic purpura (7). Recent studies have shown that beef and dairy cattle are natural reservoirs of E. coli O157:H7 (2, 43) and that E. coli O157:H7 can be isolated from the feces of asymptomatic cattle (2, 22), from raw milk (2, 9), and from poultry, pork, and lamb (12, 30). However, undercooked ground beef is the major source of E. coli O157:H7 in food-borne outbreaks (8). The E. coli O157:H7 serotype has traditionally been identified by agglutination tests (14, 21) or immunofluorescence assays (25) with polyclonal antibodies. However, use of polyclonal antisera may result in false-positive identification of E. coli O157 (3, 20). For example, polyclonal anti-O157 E. coli antisera may cross-react with E. coli O7 and O116 (13, 27). In addition, other bacteria possess cross-reacting epitopes which mimic epitopes on the lipopolysaccharide (LPS) of E. coli

Evaluation of the ‘GeneDisc’ real-time PCR system for detection of enterohaemorrhagicEscherichia coli(EHEC) O26, O103, O111, O145 and O157 strains according to their virulence markers and their O- and H-antigen-associated genes

“…Screening for the rfbE O157 gene was found more specific than O-serotyping, as cross-reactions described for Salmonella O30, Y. enterocolitica O9, Escherichia hermanii and Cit. freundii strains with an O157 antigen (Westerman et al 1997;Samuel et al 2004) were not found at the DNA-level. Similar findings were made for E. coli O111 group and the serologically cross-reacting Salmonella O35.…”

“…These tested all negative as well as all strains belonging to other types of Enterobacteriacea. We have tested other enterobacteriaceal strains that were reported to have similarities in their O-antigen to E. coli O157 such as Salmonella O30 (n = 3), and one each of Yersinia enterocolitica O9, Escherichia hermannii and Citrobacter freundii serologically positive for the E. coli O157 antigen (Westerman et al 1997;Samuel et al 2004). All these tested negative for rfbE O157 gene indicating that the detection system is specific for the E. coli rfbE O157 gene.…”

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

“…Monoclonal antibodies for the LPS of E. coli O157 have been described by two groups (Perry et al . 1988; Westerman et al . 1997).…”

“…(1988) did not present any results concerning cross reactions with bacteria that mimic the LPS of E. coli O157 on the MAbs that they developed. Those MAbs produced by Westerman et al . (1997) did not bind to the LPS of Y. enterocolitica O9 or Cit.…”

“…freundii , but did bind to strains of group N Salmonella . Of the bacteria with related LPS that were examined the only differences between the MAbs produced by Westerman et al . (1997) and the present study were the binding of 7A6 to Cit.…”