Preparation of a monoclonal antibody against Mycobacterium bovis CFP-10 and establishment and application of a competitive ELISA for bovine tuberculosis detection

Abstract:

[Background] Bovine tuberculosis is a zoonotic infectious disease mainly caused by Mycobacterium bovis, which hinders the development of breeding industry and causes huge economic burden worldwide. [Objective] To prepare monoclonal antibodies against Mycobacterium tuberculosis CFP-10 protein and establish a competitive ELISA for the detection of bovine tuberculosis. [Methods] With rHis-CFP-10 protein as the immunogen, the mouse hybridoma fusion technique was employed to establish stable hybridoma cells that secreted antibodies. A monoclonal antibody was obtained from the hybridoma cell line 8E6 and subsequently conjugated with horseradish peroxidase after purification. A competitive ELISA for the detection of bovine tuberculosis was established based on the anti-CFP-10 monoclonal antibody. To assess the applicability of the established method, we employed the commercial BOVIGAM^TM kit and the established competitive ELISA assay to detect 155 dairy cows. [Results] A hybridoma cell line 8E6 stably secreting specific antibodies against CFP-10 was obtained. A competitive ELISA method was established based on the HRP-8E6 antibody. The working concentration of the coating antigen CFP-10-ESAT-6 and the dilution factor of HRP-8E6 were 0.75 μg/mL and 1:8 000, respectively. The established method showed the Cut-off of 47.10%, the limit of detection of 0.800 μg/mL, and good reproducibility (inter and intra-batch coefficients of variance being below 10%). The established method showed the sensitivity of 54.55% and the specificity of 100.00% in the detection of the serum samples from cattle with bovine tuberculosis and healthy cattle. In the clinical trial, the established method showed a positive coincidence rate of 78.57%, a negative coincidence rate of 82.35%, and a total coincidence rate of 80.65% compared with the BOVIGAM^TM kit. [Conclusion] An anti-CFP-10 antibody was successfully prepared and a competitive ELISA method was established using a CFP-10-ESAT-6 fusion protein with high specificity. The method is accurate and reliable and can be preliminarily applied to the detection of bovine tuberculosis.

Key words:Mycobacterium bovis;monoclonal antibody;competitive ELISA;CFP-10