~ 294 ~ The Pharma Innovation Journal 2019; 8(3): 294-298 ISSN (E): 2277- 7695 ISSN (P): 2349-8242 NAAS Rating: 5.03 TPI 2019; 8(3): 294-298 © 2019 TPI www.thepharmajournal.com Received: 19-01-2019 Accepted: 24-02-2019 Gokul Chand Division of Veterinary Biotechnology, Indian Veterinary Research Institute. Izatnagar, India Deepak Kumar Division of Veterinary Biotechnology, Indian Veterinary Research Institute. Izatnagar, India PP Goswami Division of Veterinary Biotechnology, Indian Veterinary Research Institute. Izatnagar, India NS Prasad Division of Veterinary Biotechnology, Indian Veterinary Research Institute. Izatnagar, India Correspondence Gokul Chand Division of Veterinary Biotechnology, Indian Veterinary Research Institute. Izatnagar, India Recombinant fusion 24.4 kDa protein based in house ELISA for serodiagnosis of paratuberculosis Gokul Chand, Deepak Kumar, PP Goswami and NS Prasad Abstract Paratuberculosis is a zoonotic chronic infectious enteric disease of ruminants caused by Mycobacterium avium subsp. paratuberculosis. ELISA based assay is ideal and sensitive for the screening of sera from infected animals. In this study, we use Map recombinant fusion 24.4 kDa protein developed by fusing the epitopic region of 300bp each from locus tag Map 0862 and locus tag Map 1637 to produce an in-house ELISA. The recombinant fusion 24.4 kDa was purified by single step chromatography using Ni-NTA Agarose. The recombinant fusion 24.4 kDa was reacted with anti- recombinant fusion 24.4 kDa antibodies as well as bovine and sheep sera infected with Map on western blot. A total of 106 animals samples were screened by commercial kit followed by Map recombinant fusion 24.4 kDa protein-based in-house ELISA. In-house ELISA using well-characterized sera (n=106) sample from sheep and bovine yielded a sensitivity of 85% and the specificity of 84.62%. The recombinant fusion 24.4 kDa used in the present study will prove useful as a diagnostic reagent in an in-house ELISA test. Keywords: 24.4. kDa, Mycobacterium avium subsp. paratuberculosis, ELISA Introduction Paratuberculosis (Johne’s disease) is bacterial zoonotic disease widespread throughout the world caused by an intracellular pathogen Mycobacterium avium subspecies paratuberculosis (Map). It is a chronic enteric disease of domestic and wild ruminants including primates (Chiodini, 1984) [4] . Paratuberculosis is an enzootic disease on the B list of the Office des International Epizootes (OIE). The zoonotic potential of Map is not fully understood. It was shown that the Map is more readily isolated from Chron’s disease patients (Waddle et al., 2015) [19] . The incomplete understanding of the host immune response against this pathogen has hindered the development of an effective vaccine. The dairy industry incurs substantial economic losses due to reduced milk production, premature culling and reduced slaughter value (Stabel et al., 2014) [17] . It is estimated that 68.0% of US dairy herds are infected with JD, costing between $200 million to $1.5 billion per year to the dairy industry (Sohal et al., 2015) [15] . ELISA is a more sensitive and specific test for serum antibodies than any other serological tests and it has been widely used for screening herds. Since the sequencing and analysis of the entire Map genome were obtained (Leroy et al., 2005) [12] , several specific proteins have been detected in the genome of Map and the immunoreactivity of these proteins investigated (Hughes et al., 2008) [10] . Bannantine et al. (2008) [1] developed a spot protein array for initial antigen screening. EV-ELISA based on surface antigens of Map identified 96.6% of the low fecal shedders and 100% of the midlevel and high-level shedders (Eda et al., 2006) [7] . JTC ELISA based on antigens secreted in early to mid-log phase worked effectively both on serum and milk and showed a higher diagnostic sensitivity and detected low-level fecal shedders of Map (40%) (Shin et al., 2008) [14] . The studies from experimentally infected cattle on 70th,194th, and 321th-day post-infection revealed that the proteins encoded by Map 0862, Map 1087 and Map 1204 elicited strong humoral immune responses as early on 70th-day post infection antibodies against these three proteins were detected in the sera from naturally infected animals. However, the antibody titer against the protein encoded by Map 0862 reduced with the progression of time (Bannantine et al., 2008) [2] . However, individual antigens are able to identify only a subset of paratuberculosis-infected animals. Then, a mix of antigens or a recombinant protein generated after fusing epitopic regions from the two individual proteins could be a good candidate for serological diagnosis.