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Journal of Clinical Microbiology, November 2004, p. 5087-5093, Vol. 42, No. 11
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.11.5087-5093.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Development and Evaluation of an Enzyme-Linked Immunosorbent Assay for Quantifying Antibodies to Japanese Encephalitis Virus Nonstructural 1 Protein To Detect Subclinical Infections in Vaccinated Horses

Eiji Konishi,^1* Mizue Shoda,¹ Naoko Ajiro,^1, and Takashi Kondo²

Department of Health Sciences, Kobe University School of Medicine, Kobe,¹ Epizootic Research Center, Equine Research Institute, Japan Racing Association, Tochigi, Japan²

Received 4 March 2004/ Accepted 2 July 2004

Antibodies to Japanese encephalitis virus (JEV) nonstructural 1 (NS1) protein constitute a marker of natural JEV infection among populations vaccinated with inactivated JE vaccine. In Japan, with few recent human JE cases, the natural infection rate is critical to evaluate the necessity of continuing JE vaccination. A sensitive immunochemical staining method for detecting NS1 antibodies in individuals naturally and subclinically infected with JEV was previously established. Here, an enzyme-linked immunosorbent assay (ELISA) to detect NS1 antibodies in equine sera was developed and evaluated as an alternative to immunostaining. By this method, NS1 antigens contained in culture fluids from cells stably transfected with the NS1 and NS2A genes were captured by a rabbit anti-NS1 polyclonal antibody. Three nanograms per well of NS1 antigen, corresponding to 1:2 to 1:8 dilutions of the culture fluid, was sufficient for testing. ELISA values were obtained by a single-serum dilution (1:100), which correlated with ELISA titers obtained by an endpoint method. Under a tentative cutoff value (0.122) statistically calculated from NS1 antibody levels of horses in an area where JEV is not endemic, a high level of qualitative agreement (85.3%) was obtained between the ELISA and immunostaining methods. A significant correlation coefficient (0.799; P < 0.001) was also obtained between the two methods. Three experimentally infected horses seroconverted no later than 13 to 23 days postinfection, whereas 4 field horses infected during an epizootic remained positive for NS1 antibodies for at least 40 weeks. Our results indicate that the ELISA used here was sufficiently sensitive to detect subclinical infections in vaccinated equine populations.

Present address: Yashiro Health and Welfare Center of Hyogo Prefecture, Yashiro 673-1431, Japan.

This article has been cited by other articles:

• Konishi, E., Kitai, Y., Kondo, T. (2008). Utilization of Complement-Dependent Cytotoxicity To Measure Low Levels of Antibodies: Application to Nonstructural Protein 1 in a Model of Japanese Encephalitis Virus. CVI 15: 88-94 [Abstract] [Full Text]  
• Kitai, Y., Shoda, M., Kondo, T., Konishi, E. (2007). Epitope-Blocking Enzyme-Linked Immunosorbent Assay To Differentiate West Nile Virus from Japanese Encephalitis Virus Infections in Equine Sera. CVI 14: 1024-1031 [Abstract] [Full Text]