Skip to main content
Journal of Clinical Microbiology logoLink to Journal of Clinical Microbiology
. 1997 Mar;35(3):679–684. doi: 10.1128/jcm.35.3.679-684.1997

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

R B Westerman 1, Y He 1, J E Keen 1, E T Littledike 1, J Kwang 1
PMCID: PMC229650  PMID: 9041412

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 non-E. coli gram-negative enteric bacteria.

Full Text

The Full Text of this article is available as a PDF (355.5 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bettelheim K. A., Evangelidis H., Pearce J. L., Sowers E., Strockbine N. A. Isolation of a Citrobacter freundii strain which carries the Escherichia coli O157 antigen. J Clin Microbiol. 1993 Mar;31(3):760–761. doi: 10.1128/jcm.31.3.760-761.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Borczyk A. A., Karmali M. A., Lior H., Duncan L. M. Bovine reservoir for verotoxin-producing Escherichia coli O157:H7. Lancet. 1987 Jan 10;1(8524):98–98. doi: 10.1016/s0140-6736(87)91928-3. [DOI] [PubMed] [Google Scholar]
  3. Bundle D. R., Cherwonogrodzky J. W., Caroff M., Perry M. B. The lipopolysaccharides of Brucella abortus and B. melitensis. Ann Inst Pasteur Microbiol. 1987 Jan-Feb;138(1):92–98. doi: 10.1016/0769-2609(87)90083-4. [DOI] [PubMed] [Google Scholar]
  4. Caroff M., Bundle D. R., Perry M. B. Structure of the O-chain of the phenol-phase soluble cellular lipopolysaccharide of Yersinia enterocolitica serotype O:9. Eur J Biochem. 1984 Feb 15;139(1):195–200. doi: 10.1111/j.1432-1033.1984.tb07994.x. [DOI] [PubMed] [Google Scholar]
  5. Clarke R. C., McEwen S. A., Gannon V. P., Lior H., Gyles C. L. Isolation of verocytotoxin-producing Escherichia coli from milk filters in south-western Ontario. Epidemiol Infect. 1989 Apr;102(2):253–260. doi: 10.1017/s0950268800029927. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Di Fabio J. L., Perry M. B., Bundle D. R. Analysis of the lipopolysaccharide of Pseudomonas maltophilia 555. Biochem Cell Biol. 1987 Nov;65(11):968–977. doi: 10.1139/o87-126. [DOI] [PubMed] [Google Scholar]
  7. Dodds K. L., Perry M. B., McDonald I. J. Electrophoretic and immunochemical study of the lipopolysaccharides produced by chemostat-grown Escherichia coli O157. J Gen Microbiol. 1987 Sep;133(9):2679–2687. doi: 10.1099/00221287-133-9-2679. [DOI] [PubMed] [Google Scholar]
  8. Doyle M. P., Schoeni J. L. Isolation of Escherichia coli O157:H7 from retail fresh meats and poultry. Appl Environ Microbiol. 1987 Oct;53(10):2394–2396. doi: 10.1128/aem.53.10.2394-2396.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Farmer J. J., 3rd, Davis B. R. H7 antiserum-sorbitol fermentation medium: a single tube screening medium for detecting Escherichia coli O157:H7 associated with hemorrhagic colitis. J Clin Microbiol. 1985 Oct;22(4):620–625. doi: 10.1128/jcm.22.4.620-625.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fujita H., Yamaguchi S., Taira T., Iino T. A simple method for the isolation of flagellar shape mutants in Salmonella. J Gen Microbiol. 1981 Jul;125(1):213–216. doi: 10.1099/00221287-125-1-213. [DOI] [PubMed] [Google Scholar]
  11. Karmali M. A. Infection by verocytotoxin-producing Escherichia coli. Clin Microbiol Rev. 1989 Jan;2(1):15–38. doi: 10.1128/cmr.2.1.15. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Karmali M. A., Petric M., Lim C., Fleming P. C., Arbus G. S., Lior H. The association between idiopathic hemolytic uremic syndrome and infection by verotoxin-producing Escherichia coli. J Infect Dis. 1985 May;151(5):775–782. doi: 10.1093/infdis/151.5.775. [DOI] [PubMed] [Google Scholar]
  13. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  14. Lior H., Borczyk A. A. False positive identifications of Escherichia coli O157. Lancet. 1987 Feb 7;1(8528):333–333. doi: 10.1016/s0140-6736(87)92065-4. [DOI] [PubMed] [Google Scholar]
  15. March S. B., Ratnam S. Sorbitol-MacConkey medium for detection of Escherichia coli O157:H7 associated with hemorrhagic colitis. J Clin Microbiol. 1986 May;23(5):869–872. doi: 10.1128/jcm.23.5.869-872.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Montenegro M. A., Bülte M., Trumpf T., Aleksić S., Reuter G., Bulling E., Helmuth R. Detection and characterization of fecal verotoxin-producing Escherichia coli from healthy cattle. J Clin Microbiol. 1990 Jun;28(6):1417–1421. doi: 10.1128/jcm.28.6.1417-1421.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Nakane P. K., Kawaoi A. Peroxidase-labeled antibody. A new method of conjugation. J Histochem Cytochem. 1974 Dec;22(12):1084–1091. doi: 10.1177/22.12.1084. [DOI] [PubMed] [Google Scholar]
  18. Neill M. A., Agosti J., Rosen H. Hemorrhagic colitis with Escherichia coli O157:H7 preceding adult hemolytic uremic syndrome. Arch Intern Med. 1985 Dec;145(12):2215–2217. [PubMed] [Google Scholar]
  19. Park C. H., Hixon D. L., Morrison W. L., Cook C. B. Rapid diagnosis of enterohemorrhagic Escherichia coli O157:H7 directly from fecal specimens using immunofluorescence stain. Am J Clin Pathol. 1994 Jan;101(1):91–94. doi: 10.1093/ajcp/101.1.91. [DOI] [PubMed] [Google Scholar]
  20. Perry M. B., Bundle D. R. Antigenic relationships of the lipopolysaccharides of Escherichia hermannii strains with those of Escherichia coli O157:H7, Brucella melitensis, and Brucella abortus. Infect Immun. 1990 May;58(5):1391–1395. doi: 10.1128/iai.58.5.1391-1395.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Perry M. B., Bundle D. R., Gidney M. A., Lior H. Identification of Escherichia coli serotype O157 strains by using a monoclonal antibody. J Clin Microbiol. 1988 Nov;26(11):2391–2394. doi: 10.1128/jcm.26.11.2391-2394.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Perry M. B., Bundle D. R., MacLean L., Perry J. A., Griffith D. W. The structure of the antigenic lipopolysaccharide O-chains produced by Salmonella urbana and Salmonella godesberg. Carbohydr Res. 1986 Nov 15;156:107–122. doi: 10.1016/s0008-6215(00)90103-4. [DOI] [PubMed] [Google Scholar]
  23. Perry M. B., MacLean L., Griffith D. W. Structure of the O-chain polysaccharide of the phenol-phase soluble lipopolysaccharide of Escherichia coli 0:157:H7. Biochem Cell Biol. 1986 Jan;64(1):21–28. doi: 10.1139/o86-004. [DOI] [PubMed] [Google Scholar]
  24. Read S. C., Gyles C. L., Clarke R. C., Lior H., McEwen S. Prevalence of verocytotoxigenic Escherichia coli in ground beef, pork, and chicken in southwestern Ontario. Epidemiol Infect. 1990 Aug;105(1):11–20. doi: 10.1017/s0950268800047592. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Riley L. W., Remis R. S., Helgerson S. D., McGee H. B., Wells J. G., Davis B. R., Hebert R. J., Olcott E. S., Johnson L. M., Hargrett N. T. Hemorrhagic colitis associated with a rare Escherichia coli serotype. N Engl J Med. 1983 Mar 24;308(12):681–685. doi: 10.1056/NEJM198303243081203. [DOI] [PubMed] [Google Scholar]
  26. Shimada T., Kosako Y., Isshiki Y., Hisatsune K. Enterohemorrhagic Escherichia coli O157:H7 possesses somatic (O) antigen identical with that of Salmonella O301. Curr Microbiol. 1992 Oct;25(4):215–217. doi: 10.1007/BF01570721. [DOI] [PubMed] [Google Scholar]
  27. Sowers E. G., Wells J. G., Strockbine N. A. Evaluation of commercial latex reagents for identification of O157 and H7 antigens of Escherichia coli. J Clin Microbiol. 1996 May;34(5):1286–1289. doi: 10.1128/jcm.34.5.1286-1289.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Stuart F. A., Corbel M. J. Identification of a serological cross-reaction between Brucella abortus and Escherichia coli 0:157. Vet Rec. 1982 Feb 27;110(9):202–203. doi: 10.1136/vr.110.9.202-a. [DOI] [PubMed] [Google Scholar]
  29. Tortorello M. L., Stewart D. S. Antibody-direct epifluorescent filter technique for rapid, direct enumeration of Escherichia coli O157:H7 in beef. Appl Environ Microbiol. 1994 Oct;60(10):3553–3559. doi: 10.1128/aem.60.10.3553-3559.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Westerman R. B., Fortner G. W., Mills K. W., Phillips R. M., Greenwood J. M. Use of monoclonal antibodies specific for the a determinant of K88 pili for detection of enterotoxigenic Escherichia coli in pigs. J Clin Microbiol. 1993 Feb;31(2):311–314. doi: 10.1128/jcm.31.2.311-314.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Westerman R. B., Mills K. W., Phillips R. M., Fortner G. W., Greenwood J. M. Predominance of the ac variant in K88-positive Escherichia coli isolates from swine. J Clin Microbiol. 1988 Jan;26(1):149–150. doi: 10.1128/jcm.26.1.149-150.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Westerman R. B., Phillips R. M., Joens L. A. Production and characterization of monoclonal antibodies specific for lipooligosaccharide of Serpulina hyodysenteriae. J Clin Microbiol. 1995 Aug;33(8):2145–2149. doi: 10.1128/jcm.33.8.2145-2149.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Willshaw G. A., Thirlwell J., Jones A. P., Parry S., Salmon R. L., Hickey M. Vero cytotoxin-producing Escherichia coli O157 in beefburgers linked to an outbreak of diarrhoea, haemorrhagic colitis and haemolytic uraemic syndrome in Britain. Lett Appl Microbiol. 1994 Nov;19(5):304–307. doi: 10.1111/j.1472-765x.1994.tb00461.x. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Clinical Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES