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. 1995 May;100(2):214–218. doi: 10.1111/j.1365-2249.1995.tb03655.x

Detection of anti-topoisomerase I antibodies using a full length human topoisomerase I recombinant protein purified from a baculovirus expression system.

J Whyte 1, W C Earnshaw 1, J J Champoux 1, L H Parker 1, L Stewart 1, N D Hall 1, N J McHugh 1
PMCID: PMC1534349  PMID: 7743657

Abstract

Topoisomerase I (topo I) is a major systemic sclerosis (SSc)-associated autoantigen. A cDNA construct encoding full length human topo I in a recombinant baculovirus transfer vector was used to infect insect cells in culture from which recombinant protein was purified. An ELISA using recombinant protein was evaluated in 340 sera including sera from 134 patients with SSc, of whom 33 had anti-topo I antibodies detected by immunodiffusion. A high yield of pure topo I of expected molecular mass and catalytic activity was obtained. The recombinant topo I ELISA was 92% sensitive and 98% specific in detecting anti-topo I antibodies which were present almost exclusively in patients with SSc. Therefore, the potential advantages of expressing human autoantigens in eukaryotic systems for diagnostic purposes were confirmed.

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Selected References

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

  1. Arnett F. C., Edworthy S. M., Bloch D. A., McShane D. J., Fries J. F., Cooper N. S., Healey L. A., Kaplan S. R., Liang M. H., Luthra H. S. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum. 1988 Mar;31(3):315–324. doi: 10.1002/art.1780310302. [DOI] [PubMed] [Google Scholar]
  2. Catoggio L. J., Bernstein R. M., Black C. M., Hughes G. R., Maddison P. J. Serological markers in progressive systemic sclerosis: clinical correlations. Ann Rheum Dis. 1983 Feb;42(1):23–27. doi: 10.1136/ard.42.1.23. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Champoux J. J., McConaughy B. L. Purification and characterization of the DNA untwisting enzyme from rat liver. Biochemistry. 1976 Oct 19;15(21):4638–4642. doi: 10.1021/bi00666a014. [DOI] [PubMed] [Google Scholar]
  4. Cram D. S., Fisicaro N., McNeilage L. J., Coppel R. L., Harrison L. C. Antibody specificities of Thai and Australian scleroderma sera with topoisomerase I recombinant fusion proteins. J Immunol. 1993 Dec 15;151(12):6872–6881. [PubMed] [Google Scholar]
  5. D'Arpa P., Machlin P. S., Ratrie H., 3rd, Rothfield N. F., Cleveland D. W., Earnshaw W. C. cDNA cloning of human DNA topoisomerase I: catalytic activity of a 67.7-kDa carboxyl-terminal fragment. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2543–2547. doi: 10.1073/pnas.85.8.2543. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. D'Arpa P., White-Cooper H., Cleveland D. W., Rothfield N. F., Earnshaw W. C. Use of molecular cloning methods to map the distribution of epitopes on topoisomerase I (Scl-70) recognized by sera of scleroderma patients. Arthritis Rheum. 1990 Oct;33(10):1501–1511. doi: 10.1002/art.1780331007. [DOI] [PubMed] [Google Scholar]
  7. Guldner H. H., Szostecki C., Vosberg H. P., Lakomek H. J., Penner E., Bautz F. A. Scl 70 autoantibodies from scleroderma patients recognize a 95 kDa protein identified as DNA topoisomerase I. Chromosoma. 1986;94(2):132–138. doi: 10.1007/BF00286991. [DOI] [PubMed] [Google Scholar]
  8. Hardin J. A. The lupus autoantigens and the pathogenesis of systemic lupus erythematosus. Arthritis Rheum. 1986 Apr;29(4):457–460. doi: 10.1002/art.1780290401. [DOI] [PubMed] [Google Scholar]
  9. Hildebrandt S., Weiner E., Senécal J. L., Noell S., Daniels L., Earnshaw W. C., Rothfield N. F. The IgG, IgM, and IgA isotypes of anti-topoisomerase I and anticentromere autoantibodies. Arthritis Rheum. 1990 May;33(5):724–727. doi: 10.1002/art.1780330515. [DOI] [PubMed] [Google Scholar]
  10. Huff J. P., Roos G., Peebles C. L., Houghten R., Sullivan K. F., Tan E. M. Insights into native epitopes of proliferating cell nuclear antigen using recombinant DNA protein products. J Exp Med. 1990 Aug 1;172(2):419–429. doi: 10.1084/jem.172.2.419. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kato T., Yamamoto K., Takeuchi H., Okubo M., Hara E., Nakada S., Oda K., Ito K., Nishioka K. Identification of a universal B cell epitope on DNA topoisomerase I, an autoantigen associated with scleroderma. Arthritis Rheum. 1993 Nov;36(11):1580–1587. doi: 10.1002/art.1780361113. [DOI] [PubMed] [Google Scholar]
  12. Kuwana M., Kaburaki J., Mimori T., Tojo T., Homma M. Autoantigenic epitopes on DNA topoisomerase I. Clinical and immunogenetic associations in systemic sclerosis. Arthritis Rheum. 1993 Oct;36(10):1406–1413. doi: 10.1002/art.1780361013. [DOI] [PubMed] [Google Scholar]
  13. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  14. McHugh N. J., Whyte J., Harvey G., Haustein U. F. Anti-topoisomerase I antibodies in silica-associated systemic sclerosis. A model for autoimmunity. Arthritis Rheum. 1994 Aug;37(8):1198–1205. doi: 10.1002/art.1780370814. [DOI] [PubMed] [Google Scholar]
  15. McNeilage L. J., Whittingham S., McHugh N., Barnett A. J. A highly conserved 72,000 dalton centromeric antigen reactive with autoantibodies from patients with progressive systemic sclerosis. J Immunol. 1986 Oct 15;137(8):2541–2547. [PubMed] [Google Scholar]
  16. Meesters T. M., Hoet M., van den Hoogen F. H., Verheijen R., Habets W. J., van Venrooij W. J. Analysis of an immunodominant epitope of topoisomerase I in patients with systemic sclerosis. Mol Biol Rep. 1992 May;16(2):117–123. doi: 10.1007/BF00419757. [DOI] [PubMed] [Google Scholar]
  17. Miller F. W., Waite K. A., Biswas T., Plotz P. H. The role of an autoantigen, histidyl-tRNA synthetase, in the induction and maintenance of autoimmunity. Proc Natl Acad Sci U S A. 1990 Dec;87(24):9933–9937. doi: 10.1073/pnas.87.24.9933. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Mukai M., Sagawa A., Atsumi T., Jodo S., Amasaki Y., Nakabayashi T., Watanabe I., Fujisaku A., Nakagawa S. 3 cases of anti-Scl-70 (topoisomerase I) antibody associated with central nervous system lupus without renal disorder. J Rheumatol. 1993 Sep;20(9):1594–1597. [PubMed] [Google Scholar]
  19. Seelig H. P., Schröter H., Ehrfeld H., Renz M. Autoantibodies against topoisomerase I detected with the natural enzyme and overlapping recombinant peptides. J Immunol Methods. 1993 Oct 15;165(2):241–252. doi: 10.1016/0022-1759(93)90350-g. [DOI] [PubMed] [Google Scholar]
  20. Shero J. H., Bordwell B., Rothfield N. F., Earnshaw W. C. High titers of autoantibodies to topoisomerase I (Scl-70) in sera from scleroderma patients. Science. 1986 Feb 14;231(4739):737–740. doi: 10.1126/science.3003910. [DOI] [PubMed] [Google Scholar]
  21. Steen V. D., Powell D. L., Medsger T. A., Jr Clinical correlations and prognosis based on serum autoantibodies in patients with systemic sclerosis. Arthritis Rheum. 1988 Feb;31(2):196–203. doi: 10.1002/art.1780310207. [DOI] [PubMed] [Google Scholar]
  22. Tan E. M. Antinuclear antibodies: diagnostic markers for autoimmune diseases and probes for cell biology. Adv Immunol. 1989;44:93–151. doi: 10.1016/s0065-2776(08)60641-0. [DOI] [PubMed] [Google Scholar]
  23. Tan E. M., Cohen A. S., Fries J. F., Masi A. T., McShane D. J., Rothfield N. F., Schaller J. G., Talal N., Winchester R. J. The 1982 revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum. 1982 Nov;25(11):1271–1277. doi: 10.1002/art.1780251101. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Van de Water J., Gershwin M. E., Leung P., Ansari A., Coppel R. L. The autoepitope of the 74-kD mitochondrial autoantigen of primary biliary cirrhosis corresponds to the functional site of dihydrolipoamide acetyltransferase. J Exp Med. 1988 Jun 1;167(6):1791–1799. doi: 10.1084/jem.167.6.1791. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Verheijen R., Van den Hoogen F., Beijer R., Richter A., Penner E., Habets W. J., van Venrooij W. J. A recombinant topoisomerase I used for autoantibody detection in sera from patients with systemic sclerosis. Clin Exp Immunol. 1990 Apr;80(1):38–43. doi: 10.1111/j.1365-2249.1990.tb06438.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Verheijen R., de Jong B. A., van Venrooij W. J. A recombinant topoisomerase I ELISA: screening for IgG, IgM and IgA anti-topo I autoantibodies in human sera. Clin Exp Immunol. 1992 Sep;89(3):456–460. doi: 10.1111/j.1365-2249.1992.tb06980.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Vázquez-Abad D., Wallace S., Senécal J. L., Joyal F., Roussin A., Earnshaw W. C., Rothfield N. Anticentromere autoantibodies. Evaluation of an ELISA using recombinant fusion protein CENP-B as antigen. Arthritis Rheum. 1994 Feb;37(2):248–252. doi: 10.1002/art.1780370214. [DOI] [PubMed] [Google Scholar]
  29. Wang J. C. DNA topoisomerases. Annu Rev Biochem. 1985;54:665–697. doi: 10.1146/annurev.bi.54.070185.003313. [DOI] [PubMed] [Google Scholar]
  30. Weiner E. S., Earnshaw W. C., Senécal J. L., Bordwell B., Johnson P., Rothfield N. F. Clinical associations of anticentromere antibodies and antibodies to topoisomerase I. A study of 355 patients. Arthritis Rheum. 1988 Mar;31(3):378–385. doi: 10.1002/art.1780310309. [DOI] [PubMed] [Google Scholar]
  31. Weiner E. S., Hildebrandt S., Senécal J. L., Daniels L., Noell S., Joyal F., Roussin A., Earnshaw W., Rothfield N. F. Prognostic significance of anticentromere antibodies and anti-topoisomerase I antibodies in Raynaud's disease. A prospective study. Arthritis Rheum. 1991 Jan;34(1):68–77. doi: 10.1002/art.1780340111. [DOI] [PubMed] [Google Scholar]

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