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. 1994 Apr;32(4):1011–1017. doi: 10.1128/jcm.32.4.1011-1017.1994

Use of a hybrid protein consisting of the variable region of the Borrelia burgdorferi flagellin and part of the 83-kDa protein as antigen for serodiagnosis of Lyme disease.

C Rasiah 1, S Rauer 1, G S Gassmann 1, A Vogt 1
PMCID: PMC267171  PMID: 8027303

Abstract

A hybrid protein consisting of the variable region of the Borrelia burgdorferi flagellin (an 18-kDa fragment) and a 59-kDa fragment (lacking the N-terminal part) of the 83-kDa protein has been constructed by genetic engineering. It was expressed as a nonfusion protein of an apparent molecular weight of 77,000 in Escherichia coli. The suitability of this new antigen for the diagnosis of Lyme disease was tested by immunoblotting; for comparison, the recombinant variable region of the flagellin, the 18-kDa fragment (p18), and the whole recombinant 83-kDa protein (p83), both expressed in E. coli, were used. A total of 120 serum samples from various stages of Lyme disease, which were positive in two serological assays, a passive hemagglutination assay and an indirect immunofluorescence assay, were tested. By indirect immunofluorescence, 74 samples were positive for immunoglobulin G (IgG) antibodies and 72 were positive for IgM antibodies. Of these serum samples, 69 of 74 (93%) contained IgG antibodies against p18 and/or p83, and IgG antibodies were detected by the hybrid protein in 67 (90%) samples. IgM antibodies against p18 and/or p83 were detected in 60 of 72 (83%) serum samples, and 57 (79%) serum samples were reactive with the hybrid protein. Twenty serum samples of patients with a history of syphilis and 40 serum samples, negative in routine B. burgdorferi serology, were tested as controls. The hybrid protein, made up of specific epitopes of an early (p18) and late (p83) antigen, is recognized by almost the same number of patient serum samples as the individual antigens.

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Selected References

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

  1. Adam T., Gassmann G. S., Rasiah C., Göbel U. B. Phenotypic and genotypic analysis of Borrelia burgdorferi isolates from various sources. Infect Immun. 1991 Aug;59(8):2579–2585. doi: 10.1128/iai.59.8.2579-2585.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Baranton G., Postic D., Saint Girons I., Boerlin P., Piffaretti J. C., Assous M., Grimont P. A. Delineation of Borrelia burgdorferi sensu stricto, Borrelia garinii sp. nov., and group VS461 associated with Lyme borreliosis. Int J Syst Bacteriol. 1992 Jul;42(3):378–383. doi: 10.1099/00207713-42-3-378. [DOI] [PubMed] [Google Scholar]
  3. Barbour A. G., Schrumpf M. E. Polymorphisms of major surface proteins of Borrelia burgdorferi. Zentralbl Bakteriol Mikrobiol Hyg A. 1986 Dec;263(1-2):83–91. doi: 10.1016/s0176-6724(86)80107-9. [DOI] [PubMed] [Google Scholar]
  4. Barbour A. G., Tessier S. L., Hayes S. F. Variation in a major surface protein of Lyme disease spirochetes. Infect Immun. 1984 Jul;45(1):94–100. doi: 10.1128/iai.45.1.94-100.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bruckbauer H. R., Preac-Mursic V., Fuchs R., Wilske B. Cross-reactive proteins of Borrelia burgdorferi. Eur J Clin Microbiol Infect Dis. 1992 Mar;11(3):224–232. doi: 10.1007/BF02098084. [DOI] [PubMed] [Google Scholar]
  6. Coleman J. L., Benach J. L. Isolation of antigenic components from the Lyme disease spirochete: their role in early diagnosis. J Infect Dis. 1987 Apr;155(4):756–765. doi: 10.1093/infdis/155.4.756. [DOI] [PubMed] [Google Scholar]
  7. Craft J. E., Fischer D. K., Shimamoto G. T., Steere A. C. Antigens of Borrelia burgdorferi recognized during Lyme disease. Appearance of a new immunoglobulin M response and expansion of the immunoglobulin G response late in the illness. J Clin Invest. 1986 Oct;78(4):934–939. doi: 10.1172/JCI112683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Ditton H. J., Neuss M., Zöller L. Evidence that Borrelia burgdorferi immunodominant proteins p100, p94 and p83 are identical. FEMS Microbiol Lett. 1992 Jul 15;73(3):217–220. doi: 10.1016/0378-1097(92)90633-y. [DOI] [PubMed] [Google Scholar]
  9. Dressler F., Whalen J. A., Reinhardt B. N., Steere A. C. Western blotting in the serodiagnosis of Lyme disease. J Infect Dis. 1993 Feb;167(2):392–400. doi: 10.1093/infdis/167.2.392. [DOI] [PubMed] [Google Scholar]
  10. Fawcett P. T., Rose C., Gibney K. M., Chase C. A., Kiehl B., Doughty R. A. Detection of antibodies to the recombinant P39 protein of Borrelia burgdorferi using enzyme immunoassay and immunoblotting. J Rheumatol. 1993 Apr;20(4):734–738. [PubMed] [Google Scholar]
  11. Fikrig E., Huguenel E. D., Berland R., Rahn D. W., Hardin J. A., Flavell R. A. Serologic diagnosis of Lyme disease using recombinant outer surface proteins A and B and flagellin. J Infect Dis. 1992 Jun;165(6):1127–1132. doi: 10.1093/infdis/165.6.1127. [DOI] [PubMed] [Google Scholar]
  12. Gassmann G. S., Jacobs E., Deutzmann R., Göbel U. B. Analysis of the Borrelia burgdorferi GeHo fla gene and antigenic characterization of its gene product. J Bacteriol. 1991 Feb;173(4):1452–1459. doi: 10.1128/jb.173.4.1452-1459.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gassmann G. S., Kramer M., Göbel U. B., Wallich R. Nucleotide sequence of a gene encoding the Borrelia burgdorferi flagellin. Nucleic Acids Res. 1989 May 11;17(9):3590–3590. doi: 10.1093/nar/17.9.3590. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Grodzicki R. L., Steere A. C. Comparison of immunoblotting and indirect enzyme-linked immunosorbent assay using different antigen preparations for diagnosing early Lyme disease. J Infect Dis. 1988 Apr;157(4):790–797. doi: 10.1093/infdis/157.4.790. [DOI] [PubMed] [Google Scholar]
  15. Hansen K., Pii K., Lebech A. M. Improved immunoglobulin M serodiagnosis in Lyme borreliosis by using a mu-capture enzyme-linked immunosorbent assay with biotinylated Borrelia burgdorferi flagella. J Clin Microbiol. 1991 Jan;29(1):166–173. doi: 10.1128/jcm.29.1.166-173.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Horton R. M., Hunt H. D., Ho S. N., Pullen J. K., Pease L. R. Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene. 1989 Apr 15;77(1):61–68. doi: 10.1016/0378-1119(89)90359-4. [DOI] [PubMed] [Google Scholar]
  17. Jiang W., Luft B. J., Schubach W., Dattwyler R. J., Gorevic P. D. Mapping the major antigenic domains of the native flagellar antigen of Borrelia burgdorferi. J Clin Microbiol. 1992 Jun;30(6):1535–1540. doi: 10.1128/jcm.30.6.1535-1540.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Jonsson M., Noppa L., Barbour A. G., Bergström S. Heterogeneity of outer membrane proteins in Borrelia burgdorferi: comparison of osp operons of three isolates of different geographic origins. Infect Immun. 1992 May;60(5):1845–1853. doi: 10.1128/iai.60.5.1845-1853.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. Luft B. J., Gorevic P. D., Jiang W., Munoz P., Dattwyler R. J. Immunologic and structural characterization of the dominant 66- to 73-kDa antigens of Borrelia burgdorferi. J Immunol. 1991 Apr 15;146(8):2776–2782. [PubMed] [Google Scholar]
  21. Luft B. J., Mudri S., Jiang W., Dattwyler R. J., Gorevic P. D., Fischer T., Munoz P., Dunn J. J., Schubach W. H. The 93-kilodalton protein of Borrelia burgdorferi: an immunodominant protoplasmic cylinder antigen. Infect Immun. 1992 Oct;60(10):4309–4321. doi: 10.1128/iai.60.10.4309-4321.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Perng G. C., LeFebvre R. B., Johnson R. C. Further characterization of a potent immunogen and the chromosomal gene encoding it in the Lyme disease agent, Borrelia burgdorferi. Infect Immun. 1991 Jun;59(6):2070–2074. doi: 10.1128/iai.59.6.2070-2074.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Prussak C. E., Almazan M. T., Tseng B. Y. Peptide production from proteins separated by sodium dodecyl-sulfate polyacrylamide gel electrophoresis. Anal Biochem. 1989 May 1;178(2):233–238. doi: 10.1016/0003-2697(89)90630-1. [DOI] [PubMed] [Google Scholar]
  24. Rasiah C., Schiltz E., Reichert J., Vogt A. Purification and characterization of a tryptic peptide of Borrelia burgdorferi flagellin, which reduces cross-reactivity in immunoblots and ELISA. J Gen Microbiol. 1992 Jan;138(1):147–154. doi: 10.1099/00221287-138-1-147. [DOI] [PubMed] [Google Scholar]
  25. Robinson J. M., Pilot-Matias T. J., Pratt S. D., Patel C. B., Bevirt T. S., Hunt J. C. Analysis of the humoral response to the flagellin protein of Borrelia burgdorferi: cloning of regions capable of differentiating Lyme disease from syphilis. J Clin Microbiol. 1993 Mar;31(3):629–635. doi: 10.1128/jcm.31.3.629-635.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Schmitz J. L., Powell C. S., Folds J. D. Comparison of seven commercial kits for detection of antibodies to Borrelia burgdorferi. Eur J Clin Microbiol Infect Dis. 1993 Jun;12(6):419–424. doi: 10.1007/BF01967435. [DOI] [PubMed] [Google Scholar]
  27. Schägger H., von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem. 1987 Nov 1;166(2):368–379. doi: 10.1016/0003-2697(87)90587-2. [DOI] [PubMed] [Google Scholar]
  28. Steere A. C., Grodzicki R. L., Kornblatt A. N., Craft J. E., Barbour A. G., Burgdorfer W., Schmid G. P., Johnson E., Malawista S. E. The spirochetal etiology of Lyme disease. N Engl J Med. 1983 Mar 31;308(13):733–740. doi: 10.1056/NEJM198303313081301. [DOI] [PubMed] [Google Scholar]
  29. Steere A. C. Lyme disease. N Engl J Med. 1989 Aug 31;321(9):586–596. doi: 10.1056/NEJM198908313210906. [DOI] [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. Wallich R., Helmes C., Schaible U. E., Lobet Y., Moter S. E., Kramer M. D., Simon M. M. Evaluation of genetic divergence among Borrelia burgdorferi isolates by use of OspA, fla, HSP60, and HSP70 gene probes. Infect Immun. 1992 Nov;60(11):4856–4866. doi: 10.1128/iai.60.11.4856-4866.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Wilske B., Fingerle V., Herzer P., Hofmann A., Lehnert G., Peters H., Pfister H. W., Preac-Mursic V., Soutschek E., Weber K. Recombinant immunoblot in the serodiagnosis of Lyme borreliosis. Comparison with indirect immunofluorescence and enzyme-linked immunosorbent assay. Med Microbiol Immunol. 1993 Nov;182(5):255–270. doi: 10.1007/BF00579624. [DOI] [PubMed] [Google Scholar]
  33. Wilske B., Preac-Mursic V., Göbel U. B., Graf B., Jauris S., Soutschek E., Schwab E., Zumstein G. An OspA serotyping system for Borrelia burgdorferi based on reactivity with monoclonal antibodies and OspA sequence analysis. J Clin Microbiol. 1993 Feb;31(2):340–350. doi: 10.1128/jcm.31.2.340-350.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Wilske B., Preac-Mursic V., Jauris S., Hofmann A., Pradel I., Soutschek E., Schwab E., Will G., Wanner G. Immunological and molecular polymorphisms of OspC, an immunodominant major outer surface protein of Borrelia burgdorferi. Infect Immun. 1993 May;61(5):2182–2191. doi: 10.1128/iai.61.5.2182-2191.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Zumstein G., Fuchs R., Hofmann A., Preac-Mursic V., Soutschek E., Wilske B. Genetic polymorphism of the gene encoding the outer surface protein A (OspA) of Borrelia burgdorferi. Med Microbiol Immunol. 1992;181(2):57–70. doi: 10.1007/BF00189424. [DOI] [PubMed] [Google Scholar]
  36. Zöller L., Burkard S., Schäfer H. Validity of western immunoblot band patterns in the serodiagnosis of Lyme borreliosis. J Clin Microbiol. 1991 Jan;29(1):174–182. doi: 10.1128/jcm.29.1.174-182.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]

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