This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Sugden, E A Articles by Stewart, R B Search for Related Content PubMed PubMed Citation Articles by Sugden, E A Articles by Stewart, R B

 Previous Article  |  Next Article 

J Clin Microbiol. 1991 August; 29(8): 1659-1664

Chromatographic purification and characterization of antigens A and D from Mycobacterium paratuberculosis and their use in enzyme-linked immunosorbent assays for diagnosis of paratuberculosis in sheep.

Agriculture Canada, Animal Diseases Research Institute, Nepean, Ottawa, Ontario, Canada.

ABSTRACT

The protein antigens A and D were purified from culture filtrates and sonic extracts of laboratory strains of Mycobacterium paratuberculosis by salt precipitation and chromatography. The characterization of antigen A is shown here, and both antigens were evaluated along with lipoarabinomannan antigen in indirect enzyme-linked immunosorbent assays (ELISA) for the serodiagnosis of ovine paratuberculosis. After anion-exchange (DEAE-5PW) and hydrophobic (phenyl-5PW) chromatography using high-performance liquid chromatography, antigen A showed a prominant band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at 31 kDa with small amounts of low-molecular-mass proteins but with no evidence of antigen D. A single precipitin arc was evident with purified antigen A in crossed immunoelectrophoresis. The determination of the N-terminal amino acid sequence showed a high degree of homology between the 31-kDa component of antigen A and antigens of the BCG85 complex of Mycobacterium bovis BCG, a total of 24 of 26 residues being identical to those of BCG85C. A prominant SDS-PAGE band at 400 kDa and a single crossed-immunoelectrophoresis arc was also evident for antigen D after gel filtration (Sephacryl S-200), anion-exchange (DEAE-Sephacel), and concanavalin A-Sepharose affinity chromatography. By ELISA, purified antigen A detected antibody in the sera of 18 of 22 paratuberculosis-infected sheep (82% sensitivity), whereas the purified antigen D detected antibody in all 22 infected animals (100% sensitivity). Combined ELISA results showed increased specificity with some loss in sensitivity.

This article has been cited by other articles:

• Janowski, R., Auerbach-Nevo, T., Weiss, M. S. (2008). Bacterioferritin from Mycobacterium smegmatis contains zinc in its di-nuclear site. Protein Sci. 17: 1138-1150 [Abstract] [Full Text]  
• Olsen, I., Reitan, L. J., Wiker, H. G. (2000). Distinct Differences in Repertoires of Low-Molecular-Mass Secreted Antigens of Mycobacterium avium Complex and Mycobacterium tuberculosis. J. Clin. Microbiol. 38: 4453-4458 [Abstract] [Full Text]  
• Olsen, I., Reitan, L. J., Holstad, G., Wiker, H. G. (2000). Alkyl Hydroperoxide Reductases C and D Are Major Antigens Constitutively Expressed by Mycobacterium avium subsp. paratuberculosis. Infect. Immun. 68: 801-808 [Abstract] [Full Text]  
• Coetsier, C., Havaux, X., Mattelard, F., Sadatte, S., Cormont, F., Buergelt, K., Limbourg, B., Latinne, D., Bazin, H., Denef, J.-F., Cocito, C. (1998). Detection of Mycobacterium avium subsp. paratuberculosis in Infected Tissues by New Species-Specific Immunohistological Procedures. CVI 5: 446-451 [Abstract] [Full Text]