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A specific and sensitive antigen capture assay for NS1 protein quantitation in Japanese encephalitis virus infection
1Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shanghai, Shanghai Institute for Biological Sciences, 411 Hefei Road, Shanghai 200025, China.
Abstract:
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 1microgml(-1) JEV NS1 protein was released in supernatants of mammalian cells infected with JEV but <10ngml(-1) was released in sera of virus-infected mice before the onset of encephalitis and death. Moreover, NS1 protein was detected at low levels (<10ngml(-1)) in 23.8% of sera and in 10.5% of CSF of patients diagnosed as IgM-positive for JEV. This quantitative test of NS1 protein is proposed for highly specific diagnosis of acute infection with JEV genotypes I to IV.
Insights
A new assay can detect Japanese encephalitis virus (JEV) NS1 protein in patient samples. This quantitative test offers a highly specific method for diagnosing acute JEV infections.
Area of Science:
- Virology
- Immunology
- Diagnostic Assay Development
Background:
- Japanese encephalitis virus (JEV) is a significant mosquito-borne flavivirus causing endemic and epidemic disease in Asia.
- Current JEV diagnostics often rely on IgM detection, as viral isolation from blood or cerebrospinal fluid (CSF) is challenging.
- The flavivirus nonstructural glycoprotein NS1 is transiently released during viral replication.
Purpose of the Study:
- To develop and validate a quantitative JEV NS1 antigen capture assay.
- To assess the utility of JEV NS1 detection in clinical samples for diagnosing acute JEV infection.
Main Methods:
- Production and purification of a soluble hexameric JEV NS1 protein using Drosophila S2 cells.
- Development of a quantitative antigen-capture assay utilizing high-affinity monoclonal antibodies (MAbs).
- Detection of JEV NS1 in cell culture supernatants, mouse sera, and human patient sera and CSF.
Main Results:
- The developed assay demonstrated a limit of detection of 0.2 ng/mL for JEV NS1.
- Significant levels of JEV NS1 (up to 1 µg/mL) were detected in mammalian cell culture supernatants.
- Low levels of NS1 (<10 ng/mL) were found in a subset of JEV IgM-positive patient sera (23.8%) and CSF (10.5%).
Conclusions:
- A quantitative JEV NS1 antigen capture assay has been successfully established.
- The assay shows promise for highly specific diagnosis of acute JEV infection across genotypes I-IV.
- NS1 detection may serve as a valuable biomarker in conjunction with existing diagnostic methods.
