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Microbiology spectrum2025; 13(10); e0093925; doi: 10.1128/spectrum.00939-25

Development and application of a NP-cELISA for the detection of nucleoprotein antibodies of equine influenza virus.

Abstract: Equine influenza (EI), caused by the equine influenza virus (EIV), is an acute respiratory disease that has become enzootic worldwide, resulting in frequent outbreaks and substantial economic losses within the equine industry. In this study, we developed a competitive enzyme-linked immunosorbent assay (NP-cELISA) for the detection of antibodies against the EIV nucleoprotein (NP). The assay was designed by coating plates with purified monoclonal antibodies (mAbs) against the NP protein, followed by simultaneous incubation of the test serum samples and HRP-NP antigen in a competitive binding reaction. Receiver operating characteristic (ROC) curve analysis demonstrated that the assay achieved 100% sensitivity and specificity. To assess the diagnostic performance of the NP-cELISA, we evaluated 119 clinical samples in parallel using the NP-cELISA, a commercially available competitive ELISA (ID.vet-cELISA), and the hemagglutination inhibition (HI) assay as the reference standard. The results indicated that the NP-cELISA showed an 87.4% concordance rate with the HI assay, outperforming the 78.2% concordance rate observed between the ID.vet-cELISA and the HI test. Additionally, in a serological surveillance study conducted using the developed NP-cELISA in China from 2021 to 2023, equine serum samples showed an average annual seroprevalence of 37.96% for EIV antibodies. In conclusion, the NP-cELISA developed in this study demonstrates significant potential as a reliable and efficient diagnostic tool for the serological detection of EI, with broad applicability in various settings. Objective: Equine influenza (EI) is a highly contagious respiratory disease that poses significant economic and health challenges to the global equine industry. Current diagnostic methods, such as hemagglutination inhibition (HI), are accurate but complex and impractical for widespread use, especially in regions like China where commercial kits are unavailable. This study developed a competitive ELISA (cELISA) for detecting EI virus antibodies, offering a simpler, faster, and more cost-effective alternative. The assay demonstrated higher concordance with HI than existing commercial kits and effectively monitored antibody responses in vaccinated horses. Additionally, it enabled the first large-scale serological survey of EI in China, providing critical insights into the virus's prevalence. This advancement supports timely disease detection and control, benefiting veterinary practices and the equine industry worldwide.
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Publication Date: 2025-08-25 PubMed ID: 40853243PubMed Central: PMC12502703DOI: 10.1128/spectrum.00939-25Google Scholar: Lookup
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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.

Overview

  • Researchers developed a new competitive ELISA test (NP-cELISA) to detect antibodies against equine influenza virus (EIV) nucleoprotein in horse serum.
  • This test offers a simpler, faster, and highly accurate alternative to traditional methods, enabling improved diagnosis and surveillance of EI infections.

Background and Significance

  • Equine influenza (EI) is a contagious respiratory disease in horses caused by the equine influenza virus (EIV), leading to frequent outbreaks and economic losses worldwide.
  • Accurate detection of antibodies against EIV is essential for disease diagnosis, vaccine evaluation, and epidemiological surveillance.
  • Existing diagnostic methods such as hemagglutination inhibition (HI) assay are highly accurate but complex, time-consuming, and not easily implemented on a wide scale, especially in regions lacking commercial kits like China.

Development of the NP-cELISA

  • The study developed a competitive enzyme-linked immunosorbent assay targeting the nucleoprotein (NP) antibody of EIV.
  • The assay protocol involved coating microtiter plates with purified monoclonal antibodies (mAbs) specific to the NP protein of EIV.
  • Test serum samples were incubated simultaneously with horseradish peroxidase (HRP)-conjugated NP antigen, allowing competition for binding sites between serum antibodies and the labeled antigen.
  • This competitive binding format ensures only serum antibodies specific to NP can inhibit the binding of the HRP-NP antigen, enabling detection via a measurable enzymatic reaction.

Performance Evaluation

  • Receiver operating characteristic (ROC) curve analysis showed the assay achieved 100% sensitivity and 100% specificity, indicating no false positives or negatives in the test population.
  • To validate diagnostic utility, 119 clinical serum samples were tested simultaneously with:
    • The newly developed NP-cELISA
    • A commercial competitive ELISA (ID.vet-cELISA)
    • The HI assay as a reference standard
  • Results demonstrated:
    • NP-cELISA had an 87.4% concordance rate with the HI assay, indicating high agreement.
    • ID.vet-cELISA showed a lower concordance rate (78.2%) with the HI assay, suggesting the NP-cELISA outperformed the commercial kit.

Application in Serological Surveillance

  • The NP-cELISA was applied in a large-scale serological survey of equine populations in China from 2021 to 2023.
  • The study found an average annual seroprevalence of 37.96% for EIV antibodies, providing valuable data on the extent of viral exposure in horses within China.
  • This represents the first extensive use of such a diagnostic tool in this region, filling a gap due to previously unavailable commercial kits.

Advantages and Implications

  • The NP-cELISA is simple, rapid, and cost-effective compared to traditional HI assays and commercial ELISA kits.
  • Its high sensitivity and specificity make it a reliable diagnostic tool for serological detection of EI.
  • It facilitates:
    • Timely diagnosis of EI outbreaks
    • Monitoring of antibody responses post-vaccination
    • Large-scale epidemiological surveillance in regions lacking commercial assays
  • Wider application of this assay can improve disease control strategies globally, benefiting veterinary medicine and the equine industry by minimizing economic losses and improving animal health.

Cite This Article

APA
Yang Y, Guo K, Xu L, Guo W, Dong M, Liu W, Li S, Zhang Z, Chu X, Wang Y, Zhang Z, Hu Z, Wang X. (2025). Development and application of a NP-cELISA for the detection of nucleoprotein antibodies of equine influenza virus. Microbiol Spectr, 13(10), e0093925. https://doi.org/10.1128/spectrum.00939-25

Publication

Microbiology spectrum
ISSN: 2165-0497
NlmUniqueID: 101634614
Country: United States
Language: English
Volume: 13
Issue: 10
Pages: e0093925
PII: e00939-25

Researcher Affiliations

Yang, Yan
  • State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, the Chinese Academy of Agricultural Sciences, Harbin, Heilongjiang, China.
Guo, Kui
  • State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, the Chinese Academy of Agricultural Sciences, Harbin, Heilongjiang, China.
Xu, Ling
  • State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, the Chinese Academy of Agricultural Sciences, Harbin, Heilongjiang, China.
Guo, Wei
  • State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, the Chinese Academy of Agricultural Sciences, Harbin, Heilongjiang, China.
Dong, Mingqi
  • Harbin Guosheng Biotechnology Co. Ltd., Harbin, China.
Liu, Wen
  • Harbin Guosheng Biotechnology Co. Ltd., Harbin, China.
Li, Shuaijie
  • State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, the Chinese Academy of Agricultural Sciences, Harbin, Heilongjiang, China.
Zhang, Zenan
  • State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, the Chinese Academy of Agricultural Sciences, Harbin, Heilongjiang, China.
Chu, Xiaoyu
  • State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, the Chinese Academy of Agricultural Sciences, Harbin, Heilongjiang, China.
Wang, Yaoxin
  • State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, the Chinese Academy of Agricultural Sciences, Harbin, Heilongjiang, China.
Zhang, Zhenyu
  • State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, the Chinese Academy of Agricultural Sciences, Harbin, Heilongjiang, China.
  • University of Wisconsin-Madison School of Medicine and Public Health, Madison, Wisconsin, USA.
Hu, Zhe
  • State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, the Chinese Academy of Agricultural Sciences, Harbin, Heilongjiang, China.
Wang, Xiaojun
  • State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, the Chinese Academy of Agricultural Sciences, Harbin, Heilongjiang, China.
  • Institute of Western Agriculture, the Chinese Academy of Agricultural Sciences, Changji, Xinjiang, China.

MeSH Terms

  • Horses
  • Animals
  • Antibodies, Viral / blood
  • Antibodies, Viral / immunology
  • Horse Diseases / diagnosis
  • Horse Diseases / virology
  • Horse Diseases / immunology
  • Enzyme-Linked Immunosorbent Assay / methods
  • Enzyme-Linked Immunosorbent Assay / veterinary
  • Orthomyxoviridae Infections / veterinary
  • Orthomyxoviridae Infections / diagnosis
  • Orthomyxoviridae Infections / virology
  • Orthomyxoviridae Infections / immunology
  • Sensitivity and Specificity
  • Influenza A Virus, H3N8 Subtype / immunology
  • Influenza A Virus, H3N8 Subtype / isolation & purification
  • Nucleoproteins / immunology
  • Antibodies, Monoclonal / immunology
  • Hemagglutination Inhibition Tests
  • ROC Curve

Grant Funding

  • IWA2023 / Tianchi Talent Introduction Plan
  • No. 2021YFD1800500 / The National Key Research and Development Program of China
  • ZZYD2023010 / Xinjiang Talent Development Fund
  • TD2022C006 / The Nature Science Foundation of Heilongjiang Province

Conflict of Interest Statement

The authors declare no conflict of interest.

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