Development and optimization of an ESAT6-ELISA-based detection system of Mycobacterium tuberculosis complex, suitable for bovine TB eradication

Abstract

Introduction: The tuberculin test is the most common method used for the detection and control of bovine tuberculosis (TB). Specificity and sensitivity of this tuberculin test is low, so it is better to perform another test in parallel for investigation of the disease. The ELISA test is user friendly, so it can be an appropriate test for performing in parallel with the tuberculin test for an accurate detection. The aim of this study is the development of an ELISA test (kit) based on proteins smaller than 10kDa (specially ESAT-6) for the detection of TB in cattle. Materials and Methods: Proteins lower than 10kDa M.W. were isolated from sonicated bacteria and were precipitated with ammonium sulfate. Protein assay was done after dialysis. Then a checkerboard was done for the determination of the best concentration of antigens and antibodies. There were 114 serum samples that were isolated; 100 cows that three continual tuberculin tests were negative and 14 serum samples were positive (in the tuberculin test). Culture and PCR were done from lymph nodes (or tissues) of tuberculin positive cows. Stability was investigated on days 0, 1, 3, 5 and 7 in 37 °C. Results: The best concentration of antigens and antibodies were acquired 200ng and 1/100 respectively. All of the 100 samples that were negative in the tuberculin tests were negative in this ELISA kit as well, but out of 14 samples that were positive in the tuberculin test, just 8 samples were positive in the ELISA kit. Meanwhile, the results of culture and PCR were compatible with the ELISA kit's results. There was a variation of less than 10% between days 0 and 7 at 37 °C that shows at least 1 year stability of this kit in refrigerator temperatures (4–8 °C). Conclusion: Performing the ELISA test based on ESAT-6 antigen shows that this test can be a suitable way for screening beside the tuberculin test for accurate detection. It is noticed that the specificity of the ELISA test was determined more than the tuberculin test, especially since the culture and PCR are gold standards. Therefore, incorrect sampling can change the specificity of the tuberculin test. The results of this kit can encourage designing of an ELISA kit for the detection of human TB