Volume 8, Issue 16 (11-2017)                   rap 2017, 8(16): 137-144 | Back to browse issues page


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Abstract:   (3371 Views)
     Tuberculosis is the most common infectious diseases. Current diagnostic methods are either slow or lack enough sensivity or specificity. Several mycobacterial antigens are involved in the complex interaction with the immune system of the host. They can be helpful for mycobacteria diagnosis. Antigen 60 (A60) is thermostable antigen found in Mycobacterium bovis and Mycobacterium tuberculosis. This antigen, are used in ELISA systems design for diagnosis of tuberculosis. The aim of this study is purification of A60 from bacterial cytoplasm and evaluating and compare its efficiency with standard human tuberculin. With filtration chromatography, A60 were purified from other bacterial components. A60 was recognized by agar gel Immnuodiffusion with anti BCG and Anti A60 antiserum, where formed immuno-percipitiongen line with anti BCG anti serum and anti A60 anti serum. Molecular weight components of the A60 was obtained using electrophoresis. By chromatography, seven fractions obtained. Dot blot analyzing showed that, cytoplasm and cell wall of BCG contained A60. This test showed that first fraction created maximum color intensity and as a result, the highest amount of A60 was in the fractions one. In agar gel immnuodiffusion, cytoplasm sample and all factions obtained from chromatography showed positive reaction with anti A60 anti serum and fraction one, had highest amount of sediment among the other factions. Molecular weight components of the A60 were identified about 35, 38, 40 and 65 kD. Results of reactions from injected A60 and standard human tuberculin showed the effectiveness of this antigen in comparison with standard human tuberculin. Detection of antibody in the serum of patients is rapid and repeatable method. A60 with 89% sensitivity and 94% specificity could be an appropriate matter for the diagnosis of tuberculosis.With this method, without any advanced and expensive equipments or materials we can achive the desired results as quickly as possible.
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Type of Study: Research | Subject: Special
Received: 2017/10/30 | Revised: 2017/11/11 | Accepted: 2017/10/30 | Published: 2017/10/30

References
1. Charpin, D., H. Herbault, M.J. Gevaudan, M. Saadjian, de P. Micco, A. Arnaud, D. Vervloet and J. Charpin. 1990. Value of ELISA using A60 antigen in the diagnosis of active pulmonary tuberculosis. Am Rev Respir Dis, 142: 380-4. [DOI:10.1164/ajrccm/142.2.380]
2. Cocito, C. 1987. Immonological properties of A60 of BCG induction of humoral and cellular immune reaction. Scand J Immunol, 25: 579-85. [DOI:10.1111/j.1365-3083.1987.tb01084.x]
3. Cocito, C. 1991. Properties of the mycobacterial antigen complex A60 and its application to the diagnosis and prognosis of TB. Chest, 100: 1687-93. [DOI:10.1378/chest.100.6.1687]
4. Cocito, C. and F. Vanlinden. 1986. Preparation and properties of antigen 60 from Mycobacterium bovis BCG. Clin Exp Immunol, 66: 262-72.
5. Cocito, C. and F. Vanlinden. 1988. Subcellular localisation and sedimentation behaviour of antigen 60 from Mycobacterium bovis BCG. Med Microbiol Immunol, 177: 15-25. [DOI:10.1007/BF00190307]
6. Coetsier, C., M.C. Baelden, M. Coene and C. Cocito. 1994. Immunlogical analysis of the companents of the antigen complex A60 of Mycobacterium bovis BCG. Clin Diagn Lab Immunol, 1: 139-144.
7. Davies, P.D.O. 1998. Clinical Tuberculosis chapman and Hall. (2nd Ed.) London, UK, 55-80 pp.
8. Dupont, C., K. Thompson, C. Heuer, B. Gicquel and A. Murray. 2005. Identification and characterization of an immunogenic 22 kDa exported protein of Mycobacterium avium subspecies paratuberculosis. J Med Microbiol, 54: 1083-92. [DOI:10.1099/jmm.0.46163-0]
9. Ferencik, M. 1993. Hand book of Immunochemistry, Chapman and Hall. (First Ed) New Delhi, Del, India, 336-341.
10. Gilot, P. and M. Coene. 1994. Thermostable macromolecular antigens from mycobacteria. Can J Microbiol, 40: 605-11. [DOI:10.1139/m94-097]
11. Guta, S., J. Casal, S. Napp, L. Saez, A. Garcia-Saenz,Perez, B. deVal, B. Romero, J. Alvarez and A. Alvarez. 2014. Epidemiological investigation of bovin tuberculosis herd breakdowns in Spain 2009/2011. PLoS One, 15: 74-81. [DOI:10.1371/journal.pone.0104383]
12. Harboe, M. and H.G. Wiker. 1992. The 38-kDa Protein of Mycobacterium tuberculosis: A Review. J Infect Dis, 166: 874-84. [DOI:10.1093/infdis/166.4.874]
13. Harris, E. and S. Angel. 1990. Protein purification methods: A practical approach; IRL Press at Oxford University Press. (First Ed) Oxford, pp: 200-310.
14. Hoeprich, P.D. 1983. Infectious disease Harper and Row. (3rd ed) Philadelfia, 122-40 pp.
15. Hubbard, R.D., C.M. Florry and C. Cocito. 1992. Immunization of mice with the Antigen A60 of Mycobacterium bovis BCG. Clin exp Immunol, 88: 129-31. [DOI:10.1111/j.1365-2249.1992.tb03051.x]
16. Kavid, N., R. Madani, S. Hosseinkhani, N. Mosavari, F. Golchinfar, T. Emam and R. Keshavarz. 2012. Evaluation of immunogenicity of purified cell wall-associated 34 kDa antigen of Mycobacterium avium subsp. paratuberculosis infection. Hybridoma, 31: 163-167. [DOI:10.1089/hyb.2011.0108]
17. Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage. T4. Nature, 227: 680-685. [DOI:10.1038/227680a0]
18. Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J Randall. 1951. Protein measurement with the Folin phenol reagent. J Biol Chem, 1: 265-75.
19. Lozano, R. 2012. Global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: a systematic analysis for the Global Burden of Disease Study 2010. Lancet, 380: 2095-128. [DOI:10.1016/S0140-6736(12)61728-0]
20. Raplay, R. and J. Walker. 2008. Molecular Biomethods Hand book. (2nd Ed) Michigan, pp: 651-60.
21. Serena, T.E. 2014. A Global Perspective on Wound Care, 13: 548-52. [DOI:10.1089/wound.2013.0460]
22. Zeinali, M., M. Jammalan, S. Ardestani and N. Mosaveri. 2009. Immunological and cytotoxicological characterization of tuberculin purifiedprotein derivative (PPD) conjugated to single-walled carbon nanotubes. Immunology Letters, 126: 48-53. [DOI:10.1016/j.imlet.2009.07.012]

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