Tehran University of Medical Sciences Iranian Journal of Microbiology 2008-3289 12 1 2020 03 03 25 31 Malihe Honarmand Kashi Department of Biology, Islamic Azad University of North Tehran Branch, Tehran, Iran Nader Mosavari Department of PPD Tuberculin, Razi Vaccine and Serum Research Institute, Agriculture Research, Education and Extension Organization, Karaj, Iran Mitra Salehi Department of Biology, Islamic Azad University of North Tehran Branch, Tehran, Iran Naheed Mojgani Department of PPD Tuberculin, Razi Vaccine and Serum Research Institute, Agriculture Research, Education and Extension Organization, Karaj, Iran 2019 07 13 2020 01 19 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. https://ijm.tums.ac.ir/index.php/ijm/article/view/2249 https://ijm.tums.ac.ir/index.php/ijm/article/download/2249/1218