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Title: MURINE MONOCLONAL ANTIBODIES SPECIFIC FOR LIPOPOLYSACCHARIDE OF ESCHERICHIACOLI O26 AND O111

Author
item Rivera Betancourt, Mildred
item Keen, James

Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/19/2000
Publication Date: N/A
Citation: N/A

Interpretive Summary: Enteropathogenic E. coli O26 & O111 cause severe diarrhea in humans & animals. E. coli O26 & O111 producing Shiga-toxin cause bloody diarrhea & hemolytic-uremic syndrome in humans & are the most important non-O157 EHEC strains. Since there are no readily available culture protocols or diagnostic reagents to isolate & identify non-O157 EHEC from food or clinical samples, the importance & prevalence of E. coli O26, O111 & other non-O157 EHEC is underestimated. To facilitate identification & detection of EPEC & EHEC O26 & O111, 2 monoclonal antibodies (MAbs 12F5 & 15C4) of the IgM & IgG3 isotype, respectively, were generated from mice immunized with E. coli O26:H11 & O111:NM whole cell lysate. MAbs' sensitivity & specificity were evaluated by ELISA reactivity with a panel of 371 bacterial isolates. These included 285 E. coli (28 O26, 25 O111, & 232 non- O26/non-O111) of various pathotypes, O:H serotypes & 86 non-E. coli (57 Sal. spp & 29 other Gram-negative bacteria). MAb 12F5 reacted strongly wit all 28 E. coli O26 & MAb 15C4 reacted with all 25 E. coli O111. The anti- O26 MAb cross-reacted with 1 of 257 non-O26 E. coli (an O-negative: non- motile), while the anti-O111 MAb did not cross-react with 260 non-O111 E. coli. MAb 15C4 also reacted with 8 of 8 Sal. O35 which was expected since the Sal. O35 & E. coli O111 O-antigens are identical. Neither MAb cross- reacted with other Sal. or non-E. coli Gram-negative isolates. Specificity of both MAbs for their relevant LPS O-antigen was confirmed by Western blots on E. coli O26, O111, & non-O26/non-O111 E. coli whole cell lysates & purified LPS. The high diagnostic accuracy of these 2 MAbs may permit their use as immunoreagents for detection of EPEC & EHEC O26 & O111 in human & animal clinical specimens or food products & as serotyping reagents.

Technical Abstract: Escherichia coli O26 and O111 are classic enteropathogenic (EPEC) and emergent enterohemorrhagic (EHEC) serotypes. Two murine monoclonal antibodies (Mab 12F5 and 15C4) reactive with the lipopolysaccharide (LPS) O-antigen of E. coli O26 and O111, respectively, were produced and characterized. MAb diagnostic sensitivity and specificity were estimated by indirect enzyme-linked immunoabsorbent assay (ELISA) reactivity with whole-cell lysate preparations from 371 Gram-negative bacteria. These included 285 E. coli strains (28 O26, 25 O111, and 232 non-O26/non-O111) of various pathotypes and O:H serotypes and 86 non-E. coli strains (57 Salmonella spp. and 29 other Gram-negative bacteria in 22 genera). MAb 12F5 reacted strongly with all E. coli O26 isolates and MAb 15C4 reacted with all E. coli O111 strains. The anti-O26 MAb cross-reacted with one of 257 non-O26 E. coli strains (an O-untypable:non-motile isolate), while the anti-O111 MAb did not cross-react with 260 non-O111 E. coli. MAb 15C4 reacted with eight of eight Salmonella O35 that was expected since the Salmonella O35 and E. coli O111 O-antigens are structurally identical. Neither MAb cross-reacted with other Salmonella or non-E. coli Gram- negative isolates. The specificity of both MAbs to their respective LPS O-antigens was confirmed by Western immunoblotting using purified LPS preparations and whole-cell lysates of E. coli O26, O111, and non-O26/O111 isolates as antigens. The high diagnostic accuracy of these two MAbs may permit their use as immunoreagents for detection and identification of EPEC and EHEC O26 and O111 in human and animal clinical specimens or food products and as serotyping reagents.