Japanese encephalitis virus (JEV) is a zoonotic pathogen transmitted primarily by Culex mosquitoes and causes severe neurological diseases in humans and animals. The main endemic areas are the Western Pacific and Southeast Asia, and its geographical distribution has expanded in recent years. The development of a diagnosis for orthoflavivirus infections is hampered by two main problems: the short duration of viremia, resulting in a narrow detection window, and severe cross-reactivity. NS1, a secreted nonstructural protein of orthoflavivirus, holds promise as a new target for overcoming these limitations. In this study, we established a highly specific and sensitive capture ELISA for the JEV NS1 protein. First, the JEV NS1 protein was successfully expressed in mammalian cells and purified using affinity chromatography. Seven mAbs recognizing JEV NS1 were generated, and the mAb 20B6 exhibited the strongest binding affinity. Based on 20B6, a capture ELISA was developed with an optimal coating concentration of 3 μg/mL and an optimal detection antibody working concentration of 0.432 μg/mL. No cross-reactivity was observed with other orthoflaviviruses (including WNV, KUNV, USUV, MVEV, SLEV, and ZIKV) or common porcine viruses. The method could effectively detect NS1 protein in cell culture medium, cell lysates, mouse tissues, and porcine serum samples from JEV-infected subjects. This study provides a solid foundation that may be further developed into an efficient and specific tool for epidemiological surveillance of Japanese encephalitis.

Keywords: Japanese encephalitis virus; NS1 protein; antigen-capture ELISA; monoclonal antibody.