Development Of Indirect ELISA For Detection Of Antibody Against Bovine Viral Diarrhea Virus By Using Tandem Epitopes Of NS3

BVD is a world-wide disease caused by BVDV. Bovine infected by BVDV showed diarrhea, acute and chronic mucosal disease, persistent infection and immunotolerance, immunosupression, pregnant cow abortion, dead fetus and abnormal fetus. Because of their reproductive system can cause the formation of lesions and persistent infection, so the disease has caused great economic losses to cattle industry of the world. Now the main measure to control and eliminate BVDV in most countries of the world is to detect the persistent animals and knock them out. Thus establishing an effective antibody test methods, combined with effective antigen detection to confirm and eradicate the persistent animals, can play a positive role to BVDV eliminate and control effectively.Non-structural protein NS3 protein of BVDV is very conservative in pestivirus, and is a immunodominance protein. In the natural infection or live attenuated vaccines in animals after immunization can generate antibodies. We selected two B cell linear epitopes of NS3 protein in this research, optimized the coding nucleic acid of the two epitopes, and constructed expression vectors which contains different number of tandem epitopes with linker (G4S)2. The recombinate protein expressed in E.coil rosetta. An BVDV antibody ELISA was established using test antigen EX6N and lots of research was done in this ELISA, such as the specificity of the method, the stability and the accuracy of test results of commercialization kits.SDS-PAGE and western blot showed that the recombinant protein expressed in E.coil can reaction with BVDV antibody positive serum. An indirect ELISA to detect antibody against BVDV was established using the optimum recombinant protein. Finally established procedures for ELISA was: the purified EX6N protein diluted to 2μg/mL with 0.01M NaOH, and coated into the ELISA plates for 14-16h in 4℃. Add 300μL blocking buffer into each well for 2h in 37℃after a wash procedure with PBST. Then pipet 100μL test Samples into the appropriate wells(dilution 1:20) and incubate in 37℃for 1h after a wash procedure with PBST. Add 100μL conjugate antibody(dilution 1:8 000) to each well after a wash procedure with PBST and incubate in 37℃for 1h after a wash procedure with PBST. Empty the contents of the wells and wash by adding 300μL of wash solution to every well. Repeat the wash 3 more times. Pipet 100μL of substrate Solution into each well and incubate for 15 minutes at room temperature then pipet 50μL Stop Solution to each well. Blank the plate reader against the Blank wells, read the optical density at 450 nm, calculate S/P. The specificity and stability of this ELISA showed well. The comparison with this method and commercial kits showed that this method can be used to BVDV antibody test, it provides an efficient auxiliary means for the prevention and control of BVDV.To survey the prevalence of bovine viral diarrhea virus in Heilongjiang Province, We used a indirect ELISA, which detected the antibody to BVDV NS3, to examine serum samples which collected from four ranches. The results showed that the percentages of the serum samples having the antibody against BVDV were 41%, 21%, 17% and 27%. The results demonstrated that the infection rate of BVDV was in high level in Heilongjiang Province.