A specific and sensitive antigen capture assay for NS1 protein quantitation in Japanese encephalitis virus infection

► Production of high amount of soluble hexameric form of NS1 glycoprotein of Japanese encephalitis virus. ► Characterization of a highly specific monoclonal antibody against a conformational epitope of NS1 protein of Japanese encephalitis virus genotypes I to IV. ► New diagnosis ELISA test highly sensitive and specific for Japanese encephalitis detection in biological samples. Japanese encephalitis virus (JEV) is a human pathogenic, mosquito-borne flavivirus that is endemic/epidemic in Asia. JEV is rarely detected or isolated from blood or cerebrospinal fluid (CSF), and detection of IgM is generally diagnostic of the infection. The flavivirus nonstructural glycoprotein NS1 is released transiently during flavivirus replication. The aim of this study was to set up a quantitative JEV NS1 antigen capture assay. A soluble hexameric form of JEV NS1 protein was produced in a stable Drosophila S2 cell clone and purified from supernatant fluids. Two IgG1 monoclonal antibodies (MAbs) with high affinity against two different epitopes of JEV NS1 antigen were used to develop an antigen-capture assay with a limit of detection of 0.2ngml−1 NS1. Up to 1μgml−1 JEV NS1 protein was released in supernatants of mammalian cells infected with JEV but