1887

Journal of Medical Microbiology logo

Volume 41, Issue 3

Purification and partial characterisation of a novel 66-kDa seroreactive protein of HRv Free

Abstract

Summary

A seroreactive protein (TB66) was purified from culture filtrate (CF) and cell sonicate (CS) of HRv by immobilised metal affinity chromatography (IMAC) on a Ni-nitrilotriacetic acid (NTA) column. The TB66 preparations obtained by IMAC contained predominantly a 66-kDa protein with a pI of 5.5 as determined by two-dimensional electrophoresis. TB66 was detected in the CF as early as 1 week of growth of HRv. The NH-terminal amino-acid sequence showed 85 % homology with the N-terminal sequence of bovine serum albumin (BSA) and 80 % homology with human serum albumin. Amino-acid analysis indicated a difference in the amino-acid content of TB66 when compared to BSA, with an abundance of acidic amino acids. A monoclonal antibody (MAb) OD4AG3, raised in this laboratory against an complex (MAC 101) sonicate cross-reacting with HRv sonicate, recognised a heat-stable and trypsin-sensitive epitope of this protein. TB66 was also recognised by MAbs IT1 and IT20 which also react with the 14-kDa antigen of the complex. Antibodies against TB66 were present in the sera of 62 of 64 patients with tuberculosis; sera from normal healthy individuals showed no significant reactivity. TB66 appears to be a predominant secretory protein of and could play an important role in the pathogenesis of this organism.

  • Received:
  • Revised:
  • Published Online:
© 1994 The Pathological Society of Great Britain and Ireland
Loading

Article metrics loading...

/content/journal/jmm/10.1099/00222615-41-3-173
1994-09-01
2024-12-08
Loading full text...

Full text loading...

/deliver/fulltext/jmm/41/3/medmicro-41-3-173.html?itemId=/content/journal/jmm/10.1099/00222615-41-3-173&mimeType=html&fmt=ahah

References

  1. Daniel TM, Janicki BW. Mycobacterial antigens: a review of their isolation, chemistry, and immunological properties. Microbiol Rev 1978; 42:84–113
     [Google Scholar]
  2. Nagai S, Matsumoto J, Nagasuga T. Specific skin-reactive protein from culture filtrate of Mycobacterium bovis BCG. Infect Immun 1981; 31:1152–1160
     [Google Scholar]
  3. Engers HD, Houba V, Bennedsen J. et al. Results of a World Health Organization-sponsored workshop to characterize antigens recognized by Mycobacterium-specific monoclonal antibodies. Infect Immun 1986; 51:718–720
     [Google Scholar]
  4. Abou-Zeid C, Smith I, Grange JM, Ratliff RL, Steele J, Rook GAW. The secreted antigens of Mycobacterium tuberculosis and their relationship to those recognized by the available antibodies. J Gen Microbiol 1988; 134:531–538
     [Google Scholar]
  5. Daniel TM, Debanne SM. The serodiagnosis of tuberculosis and other mycobacterial diseases by enzyme-linked immunosorbent assay. Am Rev Respir Dis 1987; 135:1137–1151
     [Google Scholar]
  6. Jackett PS, Bothamley GH, Batra HV, Mistry A, Young DB, Ivanyi J. Specificity of antibodies to immunodominant mycobacterial antigens in pulmonary tuberculosis. J Clin Microbiol 1988; 26:2313–2318
     [Google Scholar]
  7. Kolk AHJ, Ho ML, Klatser PRJ. et al. Production and characterization of monoclonal antibodies to Mycobacterium tuberculosis, M. bovis (BCG) and M. leprae. Clin Exp Immunol 1984; 58:511–521
     [Google Scholar]
  8. Coates ARM, Hewitt J, Allen BW, Ivanyi J, Mitchison DA. Antigenic diversity of Mycobacterium tuberculosis and Mycobacterium bovis detected by means of monoclonal antibodies. Lancet 1981; 2:167–169
     [Google Scholar]
  9. Andersen AB, Yuan Z-L, Hasløv K, Vergmann B, Bennedsen J. Interspecies reactivity of five monoclonal antibodies to Mycobacterium tuberculosis as examined by immuno-blotting and enzyme-linked immunosorbent assay. J Clin Microbiol 1986; 23:446–451
     [Google Scholar]
  10. Hochuli E, Dobeli H, Schacher A. New metal chelate adsorbent selective for proteins and peptides containing neighbouring histidine residues. J Chromatogr 1987; 411:177–184
     [Google Scholar]
  11. Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 1979; 76:4350–4354
     [Google Scholar]
  12. Navalkar RG, Deshpande RG, Khan MB, Savariar LS, Navalkar SR. Enzyme linked immunosorbent assay of a 12-kDa protein of Mycobacterium leprae with sera from leprosy patients. Int J Lepr 1992; 60:382–389
     [Google Scholar]
  13. Pal PG, Horwitz MA. Immunization with extracellular proteins of Mycobacterium tuberculosis induces cell-mediated immune responses and substantial protective immunity in a guinea pig model of pulmonary tuberculosis. Infect Immun 1992; 60:4781–4792
     [Google Scholar]
  14. Collins FM, Lamb JR, Young DB. Biological activity of protein antigens isolated form Mycobacterium tuberculosis culture filtrate. Infect Immun 1988; 56:1260–1266
     [Google Scholar]
  15. Kaushik NK, Choudary GV, Sharma P, Shah A, Venkitasubramanian TA. Serodiagnosis of tuberculosis using purified mycobacterial proteins. Indian J Chest Dis Allied Sci 1991; 33:119–128
     [Google Scholar]
  16. Wallis RS, Aide SLM, Havlir DV, Amir-Tahmasseb MH, Daniel TM, Ellner JJ. Identification of antigens of Mycobacterium tuberculosis using human monoclonal antibodies. J Clin Invest 1989; 84:214–219
     [Google Scholar]
  17. Li Q. Production and characterization of 3 mcAbs to Mycobacterium tuberculosis. Chung Hua Chieh Ho Ho Hu Hsi Tsa Chih 1991; 14:197–198
     [Google Scholar]
  18. Engers HD, Abe M, Bloom BR. et al. Results of a World Health Organization-sponsored workshop on monoclonal antibodies to Mycobacterium leprae. Infect Immun 1985; 48:603–605
     [Google Scholar]
  19. Verbon A, Hartskeerl RA, Schuitema A, Kolk AHJ, Young DB, Lathigra R. The 14,000-molecular-weight antigen of Mycobacterium tuberculosis is related to the alphacrystallin family of low-molecular-weight heat shock proteins. J Bacteriol 1992; 174:1352–1359
     [Google Scholar]
  20. Michalski WP. Resolution of three forms of superoxide dismutase by immobilised metal affinity chromatography. J Chromatogr 1992; 576:340–345
     [Google Scholar]
  21. Van Dam ME, Wuenschell GE, Arnold FH. Metal affinity precipitation of proteins. Biotechnol Appl Biochem 1989; 11:492–502
     [Google Scholar]
  22. Maisano F, Testori SA, Grandi G. Immobilized metal-ion affinity chromatography of human growth hormone. J Chromatogr 1989; 472:422–427
     [Google Scholar]
  23. Thorns CJ, Morris JA. Common epitopes between mycobacterial and certain host tissue antigens. Clin Exp Immunol 1985; 61:323–328
     [Google Scholar]
  24. Kong TH, Coates ARM, Butcher PD, Hickman CJ, Shinnick TM. Mycobacterium tuberculosis expresses two chaperonin-60 homologs. Proc Natl Acad Sci USA 1993; 90:2608–2612
     [Google Scholar]
  25. Shinnick TM. The 65-kilodalton antigen of Mycobacterium tuberculosis. J Bacteriol 1987; 169:1080–1088
     [Google Scholar]
/content/journal/jmm/10.1099/00222615-41-3-173
Loading
Purification and partial characterisation of a novel 66-kDa seroreactive protein of Mycobacterium tuberculosis H37Rv
J. Med. Microbiol. 41, 173 (1994); https://doi.org/10.1099/00222615-41-3-173
/content/journal/jmm/10.1099/00222615-41-3-173
/content/journal/jmm/10.1099/00222615-41-3-173
Loading

Data & Media loading...

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error