Development Of An Indirect ELISA For Diagnosing Of Bovine Paratuberculosis Using A Recombinant Multi-Antigen

In order to develop an indirect ELISA for diagnosing bovine paratuberculosis, the DNA fragmentsof map086 2and map2154c were amplified from the genome DNA Mycobacterium avium Subspeciesparatuberculosis K10. Then the DNA fragments of map0862 and map2154c were spliced byoverlapping extension (SOE), yielding a fusion gene map0862-2154c. This fragment was inserted intopMD18-T vectors, and then subcloned into pET32a (+) to construct a recombinant plasmidpET0862-2154c. The recombinant plasmid was transformed into Escherichia coli BL21 (DE3). Therecombinant protein was expressed with two hexohistidine tags induced by IPTG. It was purified bynickel-NTA chromatography. The immunogenicity of the protein was tested by immunoblotting. Thepurified protein was found to be immunogenic and useful for serodiagnosis.A series of tests were carried out to determine the optimal confining solution and coat situation. L₉(₃⁴) orthogonal experiments were designed for determination of the optimal dilution of antigen, sera andgoat-anti-rabbit IgG, and their reaction time. 95 sera from healthy cattle were tested in order todetermine the critical values, the S/P values above 0.42 were considered as positive, below 0.34 asnegative and between 0.42 and 0.34 as ambiguous.Sera from 90 cattle with bovine paratuberculosis and 100 cattle without bovine paratuberculosiswere tested. The results show that the sensitivity is 84.44ï¼…(76/90) and the specificity is 97ï¼…(97/100).104 sera (54 of them from IDEXX test positive cattle and 50 of them from IDEXX test negative cattle)were also tested and the results were compared with IDEXX test results. The total agreement betweenELISA and IDEXX test was 91.35ï¼….