Establishment And Preliminary Applicaiton Of ELISA Test Method For Neospora Caninum

Neosporosis is a protozoosis, which was caused by Neospora caninum of cells incattle, sheep, dogsand other animals. It is imorptant in public health and animalhusbandry. There is no effect drug and vaccines on treatment of Neospora caninum,so it is important that early, accurate diagnosis for controlling neosporosis occurrenceand spread. For the higher of specificity, sensitivity, stability, and so on. ELISA is themost widely used enzyme immumoassay technology. Therefore, in this study,NcSAG4was chosen as the surface protein for development of indirect ELISA andDot-ELISA in neosporosis in cattle for diagnosis.In the present study, the NcSAG4ORF was amplified by PCR, and construct aprokaryotic expression vector pGEX-NcSAG4and express it in Escherichia coliBL21(DE3). It was induced with IPTG, the expressed recombinant protein wasdetected as a hand of44.79kDa by SDS-PAGE. A special reaction band to anti-Neospora caninum sera was observed in Western blotting. It has shown that thisfusion protein has a effective immunogenicity. There was no cross-reactivity withanti-C.andersoni, anti-T.gondii, anti-G.lamblia and anti-E.tenella sera. It wasexpressed in the form of inclusion body, thus extracted and dissolved the inclusionbodies with Triton X-100and8M urea respectively, followed by the differentconcentrations of urea gradient dialysis to refolding inclusion bodies.To develop and compare indirect ELISA and Dot-ELISA for detection of theantibody against Neospora caninum, recombinant protein of NcSAG4protein waspurified as coating antigen. The optimal antigen concentration for coating was0.25μgeach well, and the optimal dilutions of the sera of Neospora caninum andenzyme-labeled the second antibody were1:200and1:2000respectively, blockingagent was the10%mice srea. Intro-batch and inter-batch repeatability test of theindirect ELISA was0.79-2.82%and1.98-6.56%respectively, and it showed highsensitivity of1:1600. For the Dot-ELISA, the optimal antigen concentration for coating was0.50μg each well, the optimal dilutions of the sera of Neospora caninumwas1:200, its sensitivity is1:800, and intro-batch repeatability test of the Dot-ELISAwas less than10%. Compared with IFAT method, the coincidence rate were95%and92.5%. Because of the simple and easy to observe the result, we chose theDot-ELISA detection method to detect the cattle srea sample. Therefore, in this study,we detected the180cattle srea samples in changchun environ area, the results showedthat the infection Neospora caninum rate was13.89%.thereby, it might be provide atheoretical basis for epidemiological investigation and prevention of Neosporacaninum in changchun environ area.