Abstract：Japanese encephalitis virus (JEV) is an important zoonotic pathogen theating human health, and still lacks sensitive antigen detection method currently. NS1 protein is a secreted protein expressed by JEV and is an ideal target for diagnosis of the virus. In this study, a double antibody sandwich ELISA (DAS-ELISA) method for detecting non-structural protein 1 (NS1) was established using the constructed NS1 protein monoclonal antibodies. Monoclonal antibody hybridoma cell lines (5H2 and 2F6) against different sites of the JEV NS1 protein were used to prepare ascites. After purification by Protein G, the ELISA plate was coated with 5H2 as the capture antibody, and 2F6 labeled with HRP as detection antibody. The optimized ELISA conditions showed that the optimal concentrations of capture antibody and enzyme-labeled antibody were 25 and 2.296 μg/mL, respectively. Using this method to detect serially diluted NS1 protein in concentration of 78.125~625.000 ng/mL, there was a good linear relationship between the OD450 value and the protein concentration, indicating that this method can be used for quantitative detection of NS1 protein. Detection of porcine pseudorabies virus, porcine reproductive and respiratory syndrome virus, and porcine parvovirus by this method showed negative results, indicating good specificity. The reproducibility test results showed that the repeatability within the batch was 0.7%~2.6% and the repeatability between batches was 0.6%~2.1%. Using this method, brain tissue homogenates of JEV-infected mice were detected, and NS1 protein was clearly detected during the onset of mice (Mus musculus). This study established an ELISA method for detecting JEV NS1 protein, which provides a tool for the diagnosis of the virus.
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