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Journal of Clinical Microbiology logoLink to Journal of Clinical Microbiology
. 1988 Oct;26(10):2127–2131. doi: 10.1128/jcm.26.10.2127-2131.1988

Isolation and characterization of monoclonal antibodies to Shiga-like toxin II of enterohemorrhagic Escherichia coli and use of the monoclonal antibodies in a colony enzyme-linked immunosorbent assay.

L P Perera 1, L R Marques 1, A D O'Brien 1
PMCID: PMC266830  PMID: 3053764

Abstract

The major obstacle in large-scale epidemiological investigations of the incidence of Shiga-like toxin (SLT)-producing Escherichia coli in diarrheal stools is the lack of a rapid, specific test to detect toxin. Enterohemorrhagic E. coli produces elevated levels of SLT-I, SLT-II, or both cytotoxins (also called Verotoxins). SLT-I but not SLT-II can be neutralized by antiserum to purified Shiga toxin and by monoclonal antibodies to the B subunit of SLT-I. In this study, monoclonal antibodies were generated against a crude preparation of SLT-II produced by an E. coli K-12 strain lysogenized with the 933W toxin-converting phage of enterohemorrhagic E. coli 933. Hybridoma culture supernatants were screened for anti-SLT-II antibodies by a cytotoxicity neutralization assay and by an enzyme-linked immunosorbent assay (ELISA). Of 53 ELISA-positive lines, 5 were capable of neutralizing the cytotoxicity of SLT-II but not of SLT-I, Shiga toxin, or a variant of SLT-II produced by E. coli that causes edema disease of swine. All five monoclonal antibodies immunoprecipitated the isolated A subunit of SLT-II but not the B subunit. Of these five neutralizing monoclonal antibodies, four were of the immunoglobulin M class and one belonged to the immunoglobulin G1 subclass. All five lines had kappa light chains. These neutralizing monoclonal antibodies have been used as probes in a colony ELISA to detect SLT-II-positive bacterial colonies. The colony ELISA with these monoclonal antibodies is a specific, sensitive test with potential diagnostic value.

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Selected References

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  1. Brown J. E., Griffin D. E., Rothman S. W., Doctor B. P. Purification and biological characterization of shiga toxin from Shigella dysenteriae 1. Infect Immun. 1982 Jun;36(3):996–1005. doi: 10.1128/iai.36.3.996-1005.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Burnette W. N. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. doi: 10.1016/0003-2697(81)90281-5. [DOI] [PubMed] [Google Scholar]
  3. Donohue-Rolfe A., Keusch G. T., Edson C., Thorley-Lawson D., Jacewicz M. Pathogenesis of Shigella diarrhea. IX. Simplified high yield purification of Shigella toxin and characterization of subunit composition and function by the use of subunit-specific monoclonal and polyclonal antibodies. J Exp Med. 1984 Dec 1;160(6):1767–1781. doi: 10.1084/jem.160.6.1767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Holmes R. K., Twiddy E. M. Characterization of monoclonal antibodies that react with unique and cross-reacting determinants of cholera enterotoxin and its subunits. Infect Immun. 1983 Dec;42(3):914–923. doi: 10.1128/iai.42.3.914-923.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Jackson M. P., Newland J. W., Holmes R. K., O'Brien A. D. Nucleotide sequence analysis of the structural genes for Shiga-like toxin I encoded by bacteriophage 933J from Escherichia coli. Microb Pathog. 1987 Feb;2(2):147–153. doi: 10.1016/0882-4010(87)90106-9. [DOI] [PubMed] [Google Scholar]
  6. Karch H., Heesemann J., Laufs R. Phage-associated cytotoxin production by and enteroadhesiveness of enteropathogenic Escherichia coli isolated from infants with diarrhea in West Germany. J Infect Dis. 1987 Apr;155(4):707–715. doi: 10.1093/infdis/155.4.707. [DOI] [PubMed] [Google Scholar]
  7. Karmali M. A., Petric M., Lim C., Cheung R., Arbus G. S. Sensitive method for detecting low numbers of verotoxin-producing Escherichia coli in mixed cultures by use of colony sweeps and polymyxin extraction of verotoxin. J Clin Microbiol. 1985 Oct;22(4):614–619. doi: 10.1128/jcm.22.4.614-619.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. Kashiwazaki M., Ogawa T., Isayama Y., Akaike Y., Tamura K., Sakazaki R. Detection of Vero cytotoxic strains of Escherichia coli isolated from diseased animals. Natl Inst Anim Health Q (Tokyo) 1980 Fall;20(3):116–117. [PubMed] [Google Scholar]
  10. Kessler S. W. Cell membrane antigen isolation with the staphylococcal protein A-antibody adsorbent. J Immunol. 1976 Nov;117(5 Pt 1):1482–1490. [PubMed] [Google Scholar]
  11. Kessler S. W. Rapid isolation of antigens from cells with a staphylococcal protein A-antibody adsorbent: parameters of the interaction of antibody-antigen complexes with protein A. J Immunol. 1975 Dec;115(6):1617–1624. [PubMed] [Google Scholar]
  12. 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]
  13. Levine M. M. Escherichia coli that cause diarrhea: enterotoxigenic, enteropathogenic, enteroinvasive, enterohemorrhagic, and enteroadherent. J Infect Dis. 1987 Mar;155(3):377–389. doi: 10.1093/infdis/155.3.377. [DOI] [PubMed] [Google Scholar]
  14. Marques L. R., Moore M. A., Wells J. G., Wachsmuth I. K., O'Brien A. D. Production of Shiga-like toxin by Escherichia coli. J Infect Dis. 1986 Aug;154(2):338–341. doi: 10.1093/infdis/154.2.338. [DOI] [PubMed] [Google Scholar]
  15. O'Brien A. D., Holmes R. K. Shiga and Shiga-like toxins. Microbiol Rev. 1987 Jun;51(2):206–220. doi: 10.1128/mr.51.2.206-220.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. O'Brien A. D., LaVeck G. D., Griffin D. E., Thompson M. R. Characterization of Shigella dysenteriae 1 (Shiga) toxin purified by anti-Shiga toxin affinity chromatography. Infect Immun. 1980 Oct;30(1):170–179. doi: 10.1128/iai.30.1.170-179.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. O'Brien A. D., LaVeck G. D. Purification and characterization of a Shigella dysenteriae 1-like toxin produced by Escherichia coli. Infect Immun. 1983 May;40(2):675–683. doi: 10.1128/iai.40.2.675-683.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Olsnes S., Reisbig R., Eiklid K. Subunit structure of Shigella cytotoxin. J Biol Chem. 1981 Aug 25;256(16):8732–8738. [PubMed] [Google Scholar]
  19. Sherwood D., Snodgrass D. R., O'Brien A. D. Shiga-like toxin production from Escherichia coli associated with calf diarrhoea. Vet Rec. 1985 Feb 23;116(8):217–218. doi: 10.1136/vr.116.8.217. [DOI] [PubMed] [Google Scholar]
  20. Strockbine N. A., Jackson M. P., Sung L. M., Holmes R. K., O'Brien A. D. Cloning and sequencing of the genes for Shiga toxin from Shigella dysenteriae type 1. J Bacteriol. 1988 Mar;170(3):1116–1122. doi: 10.1128/jb.170.3.1116-1122.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Strockbine N. A., Marques L. R., Holmes R. K., O'Brien A. D. Characterization of monoclonal antibodies against Shiga-like toxin from Escherichia coli. Infect Immun. 1985 Dec;50(3):695–700. doi: 10.1128/iai.50.3.695-700.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Strockbine N. A., Marques L. R., Newland J. W., Smith H. W., Holmes R. K., O'Brien A. D. Two toxin-converting phages from Escherichia coli O157:H7 strain 933 encode antigenically distinct toxins with similar biologic activities. Infect Immun. 1986 Jul;53(1):135–140. doi: 10.1128/iai.53.1.135-140.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Weinstein D. L., Jackson M. P., Samuel J. E., Holmes R. K., O'Brien A. D. Cloning and sequencing of a Shiga-like toxin type II variant from Escherichia coli strain responsible for edema disease of swine. J Bacteriol. 1988 Sep;170(9):4223–4230. doi: 10.1128/jb.170.9.4223-4230.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Yutsudo T., Nakabayashi N., Hirayama T., Takeda Y. Purification and some properties of a Vero toxin from Escherichia coli O157:H7 that is immunologically unrelated to Shiga toxin. Microb Pathog. 1987 Jul;3(1):21–30. doi: 10.1016/0882-4010(87)90034-9. [DOI] [PubMed] [Google Scholar]

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