Studies Of Double Antibody Sandwich ELISA Kit For Detection Of JEV

Porcine epidemic Japanese encephalitis is a disease caused by Japanese encephalitis virus (Japanese encephalitis virus, JEV), this disease is a zoonotic disease which widely exist from Asia to Australia, the virus is spread by biting of mosquito, it is greatly harm to human health, while the dangers of the disease for the animal breeding industry can not be ignored, each year the disease has brought huge economic losses to the farming industry in China, the study about the Japanese encephalitis virus dual-antibody sandwich ELISA kit provide technological support and protection for farming industry in china.1. Establishment of double-antibody sandwich ELISA methodIn this study, the NJ1-1JEV strain is cultured by BHK21cells and purified as animal antigen, the inactivated virus mix with Freund’s complete adjuvant or Freund’s incomplete adjuvant which is used to inject the different parts of rabbits and obtain the rabbit anti-JEV serum. The serum is purified by Acid-sulfuric acid method,in the next step the initial extract is also purified by Protein A affinity chromatography method. rabbit anti-JEV IgG purified is used as the capture antibody. The JEV-1monoclonal antibody hybridoma cell strain prepared and saved by our laboratory is injected into mouse peritoneal of BALB/C mice treated in order to obtain ascitic fluid containing monoclonal antibodies, then monoclonal antibodies is purified by Protein A affinity chromatography method which we use as another antibody source in double antibody sandwich ELISA method.Through the optimization of various reaction conditions, the selected working conditions for the reaction are followed:rabbit anti-JEV antibody was diluted for1:320; monoclonal antibodies was diluted for1:200; sheep anti-mouse HRP was diluted1:4000 and response1h; Micro titer plates was coated by5%skim milk for1h; chromogenic substrate woke for10minutes.The method was used to detect these virus known symptoms similar disease with Japanese encephalitis virus such as pseudorabies virus, PRRS virus, porcine parvovirus, the research reveal that the cross-reactivity did not significantly appear, it means that the method established by us is highly specific. Because of detection of negative samples and mathematical statistical methods to distinguish between position and negative:the sample whose OD value is less than0.3is determined as negative, the sample whose OD values is higher than0.5is judged to be positive and the sample whose OD values is between0.3and0.5is suspected to be infected. In sensitivity test the kit can be achieved when the virus content above1.004μg/ml and compare to RT-PCR, the sensitivity of the method should be improved.2. the Stability and application of this KitWe use different microtiter plates in the same batch and different batches of kits to detect the same sample. The kit established by us is used to detect the same positive samples per month. In the stability and shelf life test, the coefficient of variation in intro-group reproducibility and inter-group reproducibility are less than10%and it is consistent with the results of the control test. We found that the kit can be stored at4℃for5month. it is coincidence rate at60%by detecting the same clinical samples.it is suitable for clinical detection and it provides a practical techniques to detect the JEV in China and laid the foundation of commercialization of swine Japanese encephalitis diagnostic kit.