Detection and characterization of purified antigenic proteins from culture filtrate of Mycobacterium bovis strain AN5
,
Abstract
Background and Objectives: Bovine tuberculosis diagnosis is usually performed by various tests with specific limitations. Mycobacterium bovis culture filtrate contains antigenic proteins that could be used to improve the sensitivity of bovine tuberculosis diagnosis. The objective of this study was to identify and purify antigenic proteins from culture filtrates of M. bovis strain AN5 for use in immunological assays.
Materials and Methods: Secreted proteins were purified from the heat-treated culture filtrate of M. bovis strain AN5. Proteins were precipitated with ammonium sulfate, fractionated by Sephadex G50 chromatography. The protein concentrations and the approximate molecular weight were determined by lowry method and 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), respectively. Immunological methods, including dot-blotting and western blotting, assessed the quality of the isolated proteins.
Results: The quantity of antigenic proteins in the culture medium was measured at far more than 15% of the amount of proteins secreted into medium. Three main chromatographic fractions obtained and showed concentrations of proteins ranging from 14 to 60 μg/μl with molecular weights in the 10 to 180 kDa range. The purified antigens showed positive reactions to the infected cattle serum throughout dot-blotting. Western blotting revealed a total of 15 to 70 kDa molecular weight proteins.
Conclusion: Immunoblotting analysis made it possible to detect and recognize novel antigens that are useful for bovine tuberculosis diagnosis improvement. This is significant since non-specific reactions were not observed when we utilized serum of cattle experimentally infected with M. bovis as a polyclonal antibody.
2. Buddle BM, McCarthy AR, Ryan TJ, Pollock JM, Vordermeier HM, Hewinson RG, et al. Use of mycobacterial peptides and recombinant proteins for the diagnosis of bovine tuberculosis in skin test-positive cattle. Vet Rec 2003 15;153:615-620.
3. Meikle V, Alito A, Llera AS, Gioffré A, Peralta A, Buddle BM, et al. Identification of novel Mycobacterium bovis antigens by dissection of crude protein fractions. Clin Vaccine Immunol 2009; 16:1352-1359.
4. Nagai SA, Matsumoto JU, Nagasuga TO. Specific skin-reactive protein from culture filtrates of Mycobacterium bovis BCG. Infect Immun 1981; 31:1152-1160.
5. Coetsier C, Baelden MC, Coene M, Cocito C. Immunological analysis of the components of the antigen complex A60 of Mycobacterium bovis BCG. Clin Diagn Lab Immunol 1994; 1:139-144.
6. Nagai S, Wiker HG, Harboe M, Kinomoto M. Isolation and partial characterization of major protein antigens in the culture fluid of Mycobacterium tuberculosis. Infect Immun 1991; 59:372-382.
7. Daniel TM, Janicki BW. Mycobacterial antigens: a review of their isolation, chemistry, and immunological properties. Microbiol Rev 1978; 42:84-113.
8. Shinnick TM. The 65-kilodalton antigen of Mycobacterium tuberculosis. J Bacteriol 1987; 169:1080-1088.
9. Nagai S, Wiker HG, Harboe M, Kinomoto M. Isolation and partial characterization of major protein antigens in the culture fluid of Mycobacterium tuberculosis. Infect Immun 1991; 59:372-382.
10. Vordermeier HM, Chambers MA, Cockle PJ, Whelan AO, Simmons J, Hewinson RG. Correlation of ESAT-6-specific gamma interferon production with pathology in cattle following Mycobacterium bovis BCG vaccination against experimental bovine tuberculosis. Infect Immun 2002; 70:3026-3032.
11. Schiller I, Oesch B, Vordermeier HM, Palmer MV, Harris BN, Orloski KA, et al. Bovine tuberculosis: a review of current and emerging diagnostic techniques in view of their relevance for disease control and eradication. Transbound Emerg Dis 2010; 57:205-220.
12. Schiller I, Vordermeier HM, Waters WR, Palmer M, Thacker T, Whelan A, et al. Assessment of Mycobacterium tuberculosis OmpATb as a novel antigen for the diagnosis of bovine tuberculosis. Clin Vaccine Immunol 2009; 16:1314-1321.
13. Seibert FB. The purification and properties of the purified protein derivative of tuberculin. Am Rev Tuberc 1934; 30:713-720.
14. Seibert FB, Munday B. The chemical composition of the active principle of tuberculin: XV. Precipitated purified tuberculin protein suitable for the preparation of a standard tuberculin. Am Rev Tuberc 1932;25:724-737.
15. Bennedsen J. Surface immunogens of Mycobacterium tuberculosis demonstrated by immunofluorescence. APMIS 1970; 78B:701-710.
16. Glenchur H, Fossieck Jr BE, Silverman M. An immediate skin test for the diagnosis of active pulmonary tuberculosis. Am Rev Respir Dis 1965; 92:741-748.
17. Daniel TM, Hinz CF. Reactivity of purified proteins and polysaccharides from Mycobacterium tuberculosis in delayed skin test and cultured lymphocyte mitogenesis assays. Infect Immun 1974; 9:44-47.
18. Ciuca V, Burghelea V, Danes M, Safta VV, Casu O, Niculicioiu R. Mathematical modeling for determining the potency of bovine tuberculin purified protein derivative (P.P.D.). Med Vet 2015;9:71-75.
19. Chaparas SD, Baer H. The immunology and chemistry of tuberculin: II. Chromatography with Sephadex of the nondialyzable tuberculin-active constituents of BCG culture filtrate. Am Rev Respir Dis 1964; 89:41-48.
20. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951; 193:265-275.
21. Sumi MG, Mathai A, Sarada C, Radhakrishnan VV. Rapid diagnosis of tuberculous meningitis by a dot immunobinding assay to detect mycobacterial antigen in cerebrospinal fluid specimens. J Clin Microbiol 1999; 37:3925-3927.
22. Raju R, Suneetha S, Sagili K, Meher VC, Saraswathi V, Satyanarayana AV, et al. Diagnostic role of the antibody response to the 38kDa, 16kDa proteins and lipoarabinomannan of Mycobacterium tuberculosis. Indian J Clin Biochem 2005; 20:123-128.
23. Gazi MJ, Miah MR, Ahmed S, Sattar AN. Comparison of western blot technique with microscopy and culture for diagnosis of tuberculosis. BJMM 2007; 1:21-24.
24. Van Pinxteren LA, Ravn P, Agger EM, Pollock J, Andersen P. Diagnosis of tuberculosis based on the two specific antigens ESAT-6 and CFP10. Clin Diagn Lab Immunol 2000; 7:155-160.
25. Harboe M, Nagai S, Patarroyo ME, Torres ML, Ramirez C, Cruz N. Properties of proteins MPB64, MPB70, and MPB80 of Mycobacterium bovis BCG. Infect Immun 1986; 52:293-302.
26. Bassey EO, Life PF, Catty D, Gaston JS, Kumararatne DS. T-cell response to mycobacterial proteins: A comparative study of tuberculous and control immunoblots of Mycobacterium tuberculosis and M. bovis BCG. Tuber Lung Dis 1996; 77:146-153.
27. Lamb JR, Young DB. A novel approach to the identification of T-cell epitopes in Mycobacterium tuberculosis using human T-lymphocyte clones. Immunology 1987; 60:1-5.
28. Carr MD, Bloemink MJ, Dentten E, Whelan AO, Gordon SV, Kelly G, et al. Solution structure of the Mycobacterium tuberculosis complex protein MPB70 from tuberculosis pathogenesis to inherited human corneal disease. J Biol Chem 2003; 278:43736-43743.
29. Michell SL, Whelan AO, Wheeler PR, Panico M, Easton RL, Etienne AT, et al. The MPB83 antigen from Mycobacterium bovis contains O-linked mannose and (1→ 3)-mannobiose moieties. J Biol Chem 2003; 278:16423-16432.
30. Munk ME, Schoel B, Kaufmann SH. T cell responses of normal individuals towards recombinant protein antigens of Mycobacterium tuberculosis. Eur J Immunol 1988; 18:1835-1838.
| Files | ||
| Issue | Vol 12 No 1 (2020) | |
| Section | Original Article(s) | |
| DOI | https://doi.org/10.18502/ijm.v12i1.2513 | |
| Keywords | ||
| Mycobacterium bovis Purified antigenic proteins Dot-blot Western blot | ||
| Rights and permissions | |
|
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. |
