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. 1992 Jun;30(6):1544–1550. doi: 10.1128/jcm.30.6.1544-1550.1992

Monoclonal antibodies specific for Clostridium difficile toxin B and their use in immunoassays.

F Müller 1, C Stiegler 1, U Hadding 1
PMCID: PMC265326  PMID: 1378062

Abstract

Five mouse monoclonal antibodies (MAbs) against Clostridium difficile toxin B have been raised and characterized. Three of them were immunoglobulin M (IgM) antibodies (6B10, 6G3, and 10B9), and the other two were of the IgG1 isotype (9E5 and 17G2), recognizing specifically two distinct epitopes on the toxin B molecule. No MAb was able to neutralize cytotoxic activity significantly. The two IgG1 MAbs were purified and applied to various immunodiagnostic assays. MAbs coupled to latex beads were used for specific removal of toxin B from cytotoxic samples and for agglutination assay. An indirect sandwich enzyme-linked immunosorbent assay with MAb 9E5 or 17G2 as the capture antibody was established for identification of toxin B with a lower detection limit of 5 ng/ml.

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Selected References

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  1. Banno Y., Kobayashi T., Kono H., Watanabe K., Ueno K., Nozawa Y. Biochemical characterization and biologic actions of two toxins (D-1 and D-2) from Clostridium difficile. Rev Infect Dis. 1984 Mar-Apr;6 (Suppl 1):S11–S20. doi: 10.1093/clinids/6.supplement_1.s11. [DOI] [PubMed] [Google Scholar]
  2. Barroso L. A., Wang S. Z., Phelps C. J., Johnson J. L., Wilkins T. D. Nucleotide sequence of Clostridium difficile toxin B gene. Nucleic Acids Res. 1990 Jul 11;18(13):4004–4004. doi: 10.1093/nar/18.13.4004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  4. DiPersio J. R., Varga F. J., Conwell D. L., Kraft J. A., Kozak K. J., Willis D. H. Development of a rapid enzyme immunoassay for Clostridium difficile toxin A and its use in the diagnosis of C. difficile-associated disease. J Clin Microbiol. 1991 Dec;29(12):2724–2730. doi: 10.1128/jcm.29.12.2724-2730.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dove C. H., Wang S. Z., Price S. B., Phelps C. J., Lyerly D. M., Wilkins T. D., Johnson J. L. Molecular characterization of the Clostridium difficile toxin A gene. Infect Immun. 1990 Feb;58(2):480–488. doi: 10.1128/iai.58.2.480-488.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Däubener W., Leiser E., von Eichel-Streiber C., Hadding U. Clostridium difficile toxins A and B inhibit human immune response in vitro. Infect Immun. 1988 May;56(5):1107–1112. doi: 10.1128/iai.56.5.1107-1112.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Krishnan C. Detection of Clostridium difficile toxins by enzyme immunoassay. J Hyg (Lond) 1986 Feb;96(1):5–12. doi: 10.1017/s0022172400062471. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. Laughon B. E., Viscidi R. P., Gdovin S. L., Yolken R. H., Bartlett J. G. Enzyme immunoassays for detection of Clostridium difficile toxins A and B in fecal specimens. J Infect Dis. 1984 May;149(5):781–788. doi: 10.1093/infdis/149.5.781. [DOI] [PubMed] [Google Scholar]
  10. Levine H. G., Kennedy M., LaMont J. T. Counterimmunoelectrophoresis vs. cytotoxicity assay for the detection of Clostridium difficile toxin. J Infect Dis. 1982 Mar;145(3):398–398. doi: 10.1093/infdis/145.3.398. [DOI] [PubMed] [Google Scholar]
  11. Libby J. M., Wilkins T. D. Production of antitoxins to two toxins of Clostridium difficile and immunological comparison of the toxins by cross-neutralization studies. Infect Immun. 1982 Jan;35(1):374–376. doi: 10.1128/iai.35.1.374-376.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Lyerly D. M., Krivan H. C., Wilkins T. D. Clostridium difficile: its disease and toxins. Clin Microbiol Rev. 1988 Jan;1(1):1–18. doi: 10.1128/cmr.1.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Lyerly D. M., Phelps C. J., Toth J., Wilkins T. D. Characterization of toxins A and B of Clostridium difficile with monoclonal antibodies. Infect Immun. 1986 Oct;54(1):70–76. doi: 10.1128/iai.54.1.70-76.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Lyerly D. M., Phelps C. J., Wilkins T. D. Monoclonal and specific polyclonal antibodies for immunoassay of Clostridium difficile toxin A. J Clin Microbiol. 1985 Jan;21(1):12–14. doi: 10.1128/jcm.21.1.12-14.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Lyerly D. M., Sullivan N. M., Wilkins T. D. Enzyme-linked immunosorbent assay for Clostridium difficile toxin A. J Clin Microbiol. 1983 Jan;17(1):72–78. doi: 10.1128/jcm.17.1.72-78.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lyerly D. M., Wilkins T. D. Commercial latex test for Clostridium difficile toxin A does not detect toxin A. J Clin Microbiol. 1986 Mar;23(3):622–623. doi: 10.1128/jcm.23.3.622-623.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Meador J., 3rd, Tweten R. K. Purification and characterization of toxin B from Clostridium difficile. Infect Immun. 1988 Jul;56(7):1708–1714. doi: 10.1128/iai.56.7.1708-1714.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Nguyen V. K., Rihn B., Heckel C., Bisseret F., Girardot R., Monteil H. Enzyme immunoassay (ELISA) for detection of Clostridium difficile toxin B in specimens of faeces. J Med Microbiol. 1990 Apr;31(4):251–257. doi: 10.1099/00222615-31-4-251. [DOI] [PubMed] [Google Scholar]
  19. Rothman S. W., Gentry M. K., Brown J. E., Foret D. A., Stone M. J., Strickler M. P. Immunochemical and structural similarities in toxin A and toxin B of Clostridium difficile shown by binding to monoclonal antibodies. Toxicon. 1988;26(6):583–597. doi: 10.1016/0041-0101(88)90239-5. [DOI] [PubMed] [Google Scholar]
  20. Sauerborn M., von Eichel-Streiber C. Nucleotide sequence of Clostridium difficile toxin A. Nucleic Acids Res. 1990 Mar 25;18(6):1629–1630. doi: 10.1093/nar/18.6.1629. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Torres J. F. Purification and characterisation of toxin B from a strain of Clostridium difficile that does not produce toxin A. J Med Microbiol. 1991 Jul;35(1):40–44. doi: 10.1099/00222615-35-1-40. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Walker R. C., Ruane P. J., Rosenblatt J. E., Lyerly D. M., Gleaves C. A., Smith T. F., Pierce P. F., Jr, Wilkins T. D. Comparison of culture, cytotoxicity assays, and enzyme-linked immunosorbent assay for toxin A and toxin B in the diagnosis of Clostridium difficile-related enteric disease. Diagn Microbiol Infect Dis. 1986 May;5(1):61–69. doi: 10.1016/0732-8893(86)90092-1. [DOI] [PubMed] [Google Scholar]
  24. Woods G. L., Iwen P. C. Comparison of a dot immunobinding assay, latex agglutination, and cytotoxin assay for laboratory diagnosis of Clostridium difficile-associated diarrhea. J Clin Microbiol. 1990 May;28(5):855–857. doi: 10.1128/jcm.28.5.855-857.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. von Eichel-Streiber C., Harperath U., Bosse D., Hadding U. Purification of two high molecular weight toxins of Clostridium difficile which are antigenically related. Microb Pathog. 1987 May;2(5):307–318. doi: 10.1016/0882-4010(87)90073-8. [DOI] [PubMed] [Google Scholar]
  26. von Eichel-Streiber C., Laufenberg-Feldmann R., Sartingen S., Schulze J., Sauerborn M. Cloning of Clostridium difficile toxin B gene and demonstration of high N-terminal homology between toxin A and B. Med Microbiol Immunol. 1990;179(5):271–279. doi: 10.1007/BF00192465. [DOI] [PubMed] [Google Scholar]
  27. von Eichel-Streiber C., Suckau D., Wachter M., Hadding U. Cloning and characterization of overlapping DNA fragments of the toxin A gene of clostridium difficile. J Gen Microbiol. 1989 Jan;135(1):55–64. doi: 10.1099/00221287-135-1-55. [DOI] [PubMed] [Google Scholar]

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