Studies On The Standardization Of Ag-Capture ELISA Kit For Detecting Bovine Viral Diarrhea Virus

BVDV was reproduced in MDBK cells, and the viruses were collected. The viruses were concentrated and purified by PEG and high-speed centrifugation through sucrose cushions. The rabbits were artificially infected with the Purified viruses, and the results showed the viruses didn't lead to the conventional rabbit fever reaction and were attenuated.Chickens and rabbits were immunized by purified BVDV and hyperimmune serum whose AGP titer was more than 1:32 and IgY whose ELISA titer was 1:6400of anti-BVDV were prepared, and get development law of its. In the same time, seven strains hybridized tumor cells could secret antibodies after anabiosis.Conditions were optimized for Ag-C-ELISA, and the diagnostic ways were successfully standardized and then two kits were developed. The result shown that ELISA plates from BBI are suitable. The optimal concentration of hyperimmune serum and IgY of anti-BVDV for coating were 1:1000 and 1:50, serum specimens which were being detected was 1:20, McAb was 1:10, HRP-goat anti mouse IgG was 1:800. The lowest detective concentration of these kits was 274ng/mL and compared with AGP, the positive coincidence rat of the kits was 100% and the sensitivity of the kits was 100 times higher than AGP. It was confirmed that there were no cross-reaction between Rotavirus and Bovine coronavirus, but there was with CSFV. Through quality control, the value of P/N was can meet the standards of analogic method. It was showed that the kits held excellent stability .It was stable well when storing 4℃for one month.Finally, it is highly specificity, sensitivity and repeatability to detecting BVDV antigen with two diagnostic kits, so they can be widely applied and can provide a kind of effective measure to detect BVDV and is a good base to further develop and merchandise the kits.