Designing of Indirect ELISA system using secreted antigens of Mycobacterium avium subsp. paratuberculosis for Diagnosis of paratuberculosis

Background and Aim: Mycobacterium avium subsp. paratubercuiosis (MAP) is the causative agent of paratuberculosis, also called as Johne's disease and is considered as the cause of irrecoverable economic losses in livestock industry. For the detection of the paratuberculosis, indirect ELISA has been highly considered as a simple method with high sensitivity and specificity. Accordingly, this study aims at designing a system of indirect ELISA for the detection of paratuberculosis. Materials and Methods: A total of ۱۰۰ serum samples from ۱۰ herds, in Tehran and Alborz provinces in ۲۰۱۵, in which paratuberculosis has been proven by culture, were selected and surveyed using the standard kit and the internal system was designed according to the standard kit. To design ELISA system, by using secretory antigens and confirmed positive and negative serum samples were used and checkerboard titration was performed. To determine the cutoff point, the results of the commercial kit were used as gold standard. Results: According to the commercial ELISA kit results (۱۵ positive samples and ۸۵ negative samples), the best concentration of antigen and antibody dilution were evaluated as ۱.۲ µg and ۱.۱۰۰ per well, respectively. Furthermore, the cutoff point was determined as ۰.۴۴. The sensitivity and specificity were evaluated as ۷۰% and ۱۰۰%, respectively. Conclusions: Secreted antigens in M. avium subsp. paratubercuiosis are sensitive to detect the infected animals but it is difficult to detect bacteria from feces in the early stages of disease. Therefore, by using indirect designed ELISA, it can be detected antibodies in the early stages of the disease.