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Home / Equine Research Bank / J Clin Microbiol / Development and evaluation of an enzyme-linked immunosorbent...
Journal of clinical microbiology2004; 42(11); 5087-5093; doi: 10.1128/JCM.42.11.5087-5093.2004

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.

Abstract: 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.
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Publication Date: 2004-11-06 PubMed ID: 15528700PubMed Central: PMC525176DOI: 10.1128/JCM.42.11.5087-5093.2004Google Scholar: Lookup
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  • Journal Article
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  • Non-U.S. Gov't

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

This research article presents an improved method for detecting antibodies linked to Japanese Encephalitis Virus (JEV) in horses. Using an enzyme-linked immunosorbent assay (ELISA), the researchers have found a more sensitive and efficient way to monitor natural and subclinical infections in horses vaccinated against JEV.

Development of the Assay Method

  • The objective of this research was to develop an enzyme-linked immunosorbent assay (ELISA) to detect nonstructural 1 (NS1) antibodies in horse sera. These antibodies are associated with natural JEV infections, specifically in populations vaccinated with an inactivated JE vaccine.
  • To develop 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. The NS1 antigen measure used was three nanograms per well, which corresponds to 1:2 to 1:8 dilutions of the culture fluid.
  • Using a single-serum dilution, ELISA values were obtained which were shown to correlate with ELISA titers derived from an endpoint method.

Evaluation of the ELISA Method

  • The performance of the ELISA method was then evaluated against immunostaining, an existing technique of detecting NS1 antibodies.
  • A tentative cut-off value calculated from NS1 antibody levels in horses from an area where JEV is not endemic was used. The ELISA method showed a high level of qualitative agreement (85.3%) with the immunostaining method.
  • A statistically significant correlation coefficient was also obtained between these two methods, indicating that the ELISA method was reliably sensitive.

Experimental Validation and Potential Applications

  • The ELISA technique was validated using experimental and field samples. Three experimentally infected horses seroconverted (showed the presence of specific antibodies in the blood serum as a response to infection) between 13-23 days after infection. Additionally, four field horses infected during an epizootic (disease event in an animal population) tested positive for NS1 antibodies for at least 40 weeks, demonstrating long-lasting response to natural infection.
  • The results of this study indicate that the developed ELISA method is sufficiently sensitive to detect subclinical infections in vaccinated equine populations. It could hence be an effective tool for monitoring the rate of natural JEV infections among vaccinated horse populations, aiding in evaluating the need for continued vaccination in places like Japan, where human JE cases have been low recently.

Cite This Article

APA
Konishi E, Shoda M, Ajiro N, Kondo T. (2004). 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. J Clin Microbiol, 42(11), 5087-5093. https://doi.org/10.1128/JCM.42.11.5087-5093.2004

Publication

Journal of clinical microbiology
ISSN: 0095-1137
NlmUniqueID: 7505564
Country: United States
Language: English
Volume: 42
Issue: 11
Pages: 5087-5093

Researcher Affiliations

Konishi, Eiji
  • Department of Health Sciences, Kobe University School of Medicine, Suma-ku, Kobe 654-0142, Japan. ekon@ams.kobe-u.ac.jp
Shoda, Mizue
    Ajiro, Naoko
      Kondo, Takashi

        MeSH Terms

        • Animals
        • Antibodies, Viral / blood
        • Encephalitis Virus, Japanese / immunology
        • Encephalitis, Japanese / immunology
        • Encephalitis, Japanese / prevention & control
        • Encephalitis, Japanese / veterinary
        • Encephalitis, Japanese / virology
        • Enzyme-Linked Immunosorbent Assay
        • Horse Diseases / diagnosis
        • Horse Diseases / immunology
        • Horse Diseases / prevention & control
        • Horse Diseases / virology
        • Horses
        • Japanese Encephalitis Vaccines / administration & dosage
        • Japanese Encephalitis Vaccines / immunology
        • Rabbits
        • Reproducibility of Results
        • Sensitivity and Specificity
        • Vaccination
        • Viral Nonstructural Proteins / immunology

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