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 tuber- culosis 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. Pro- teins 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, as- sessed 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 pro- teins 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 tu- berculosis 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.