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. 1992 Jun;60(6):2488–2492. doi: 10.1128/iai.60.6.2488-2492.1992

Localization of two epitopes recognized by monoclonal antibody PCG-4 on Clostridium difficile toxin A.

S M Frey 1, T D Wilkins 1
PMCID: PMC257186  PMID: 1375199

Abstract

The toxin A gene of Clostridium difficile contains a 2.5-kb region encoding a series of contiguous repeating units located at the COOH terminus of the molecule. We previously showed that the monoclonal antibody (MAb) PCG-4, which neutralizes the enterotoxic activity of toxin A, binds to epitopes located within these repeating units. In the present study, we subcloned a series of fragments from this portion of the gene. The recombinant peptides expressed from the gene fragments were examined for reactivity with MAb PCG-4 to identify the epitopes involved in binding. Our results showed that MAb PCG-4 recognizes epitopes in amino acid residues 2097 through 2141 and amino acid residues 2355 through 2398.

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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. Bartlett J. G., Chang T. W., Gurwith M., Gorbach S. L., Onderdonk A. B. Antibiotic-associated pseudomembranous colitis due to toxin-producing clostridia. N Engl J Med. 1978 Mar 9;298(10):531–534. doi: 10.1056/NEJM197803092981003. [DOI] [PubMed] [Google Scholar]
  4. Birnboim H. C. A rapid alkaline extraction method for the isolation of plasmid DNA. Methods Enzymol. 1983;100:243–255. doi: 10.1016/0076-6879(83)00059-2. [DOI] [PubMed] [Google Scholar]
  5. 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: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  6. Chou P. Y., Fasman G. D. Empirical predictions of protein conformation. Annu Rev Biochem. 1978;47:251–276. doi: 10.1146/annurev.bi.47.070178.001343. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. George R. H., Symonds J. M., Dimock F., Brown J. D., Arabi Y., Shinagawa N., Keighley M. R., Alexander-Williams J., Burdon D. W. Identification of Clostridium difficile as a cause of pseudomembranous colitis. Br Med J. 1978 Mar 18;1(6114):695–695. doi: 10.1136/bmj.1.6114.695. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. doi: 10.1016/s0022-2836(83)80284-8. [DOI] [PubMed] [Google Scholar]
  10. He M., Beechey R. B., Kaderbhai M. A. A procedure for electro-elution of DNA from agarose gels. Biotechniques. 1988 Sep;6(8):728–730. [PubMed] [Google Scholar]
  11. Helfman D. M., Feramisco J. R., Fiddes J. C., Thomas G. P., Hughes S. H. Identification of clones that encode chicken tropomyosin by direct immunological screening of a cDNA expression library. Proc Natl Acad Sci U S A. 1983 Jan;80(1):31–35. doi: 10.1073/pnas.80.1.31. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Krivan H. C., Clark G. F., Smith D. F., Wilkins T. D. Cell surface binding site for Clostridium difficile enterotoxin: evidence for a glycoconjugate containing the sequence Gal alpha 1-3Gal beta 1-4GlcNAc. Infect Immun. 1986 Sep;53(3):573–581. doi: 10.1128/iai.53.3.573-581.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Larson H. E., Price A. B., Honour P., Borriello S. P. Clostridium difficile and the aetiology of pseudomembranous colitis. Lancet. 1978 May 20;1(8073):1063–1066. doi: 10.1016/s0140-6736(78)90912-1. [DOI] [PubMed] [Google Scholar]
  14. Libby J. M., Jortner B. S., Wilkins T. D. Effects of the two toxins of Clostridium difficile in antibiotic-associated cecitis in hamsters. Infect Immun. 1982 May;36(2):822–829. doi: 10.1128/iai.36.2.822-829.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Lyerly D. M., Lockwood D. E., Richardson S. H., Wilkins T. D. Biological activities of toxins A and B of Clostridium difficile. Infect Immun. 1982 Mar;35(3):1147–1150. doi: 10.1128/iai.35.3.1147-1150.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. 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]
  19. 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]
  20. Martinez R. D., Wilkins T. D. Purification and characterization of Clostridium sordellii hemorrhagic toxin and cross-reactivity with Clostridium difficile toxin A (enterotoxin). Infect Immun. 1988 May;56(5):1215–1221. doi: 10.1128/iai.56.5.1215-1221.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Sullivan N. M., Pellett S., Wilkins T. D. Purification and characterization of toxins A and B of Clostridium difficile. Infect Immun. 1982 Mar;35(3):1032–1040. doi: 10.1128/iai.35.3.1032-1040.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Taylor N. S., Thorne G. M., Bartlett J. G. Comparison of two toxins produced by Clostridium difficile. Infect Immun. 1981 Dec;34(3):1036–1043. doi: 10.1128/iai.34.3.1036-1043.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]

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