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Pathogens (Basel, Switzerland)2025; 14(6); 575; doi: 10.3390/pathogens14060575

Comparative Evaluation of a Multistrain Indirect ELISA Targeting Anti- p26 and gp45 Antibodies for EIAV Detection.

Abstract: Equine Infectious Anemia Virus (EIAV), a lentivirus marked by considerable genetic variability, poses significant diagnostic challenges. Existing diagnostic tools encompass the Agar Gel Immunodiffusion Assay (AGID), enzyme-linked immunosorbent assay (ELISA), and Western blotting (WB). ELISA and AGID mainly utilize the p26 capsid protein, often sourced from the Wyoming reference strain. To broaden the range of viral proteins and strains employed in these immunoassays, we previously developed a novel p26/double-strain gp45 indirect ELISA. In this study, we evaluated the performance of this ELISA in comparison to two commercial EIAV ELISAs using Cohen's Kappa test and Bayesian Latent Class Analysis (BLCA), a statistical method that estimates test performance without requiring a perfect reference standard. A comparison with the official classification of the sera by the Italian Veterinary Service was also performed. A total of 372 serum samples, including 96 that were positives by all three tests, were analyzed. Results from both Cohen's Kappa test and BLCA, alongside comparison with official classifications, affirm the diagnostic reliability of the two commercial ELISAs and suggest that the novel ELISA, with its enhanced antigenic diversity, could offer an accurate and reliable diagnostic option for EIAV. This novel assay enhances existing commercial ELISAs and has the potential to strengthen routine diagnostic workflows.
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Publication Date: 2025-06-08 PubMed ID: 40559583PubMed Central: PMC12196156DOI: 10.3390/pathogens14060575Google Scholar: Lookup
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  • Journal Article
  • Comparative Study
  • Evaluation Study

Summary

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Overview

  • This study evaluates a newly developed multistrain indirect ELISA test for detecting Equine Infectious Anemia Virus (EIAV) antibodies by comparing it against two commercial ELISA kits and existing official diagnostic methods.
  • The research explores whether the inclusion of multiple viral proteins and strains improves diagnostic accuracy for EIAV, a virus known for its genetic variability.

Introduction to EIAV and Diagnostic Challenges

  • Equine Infectious Anemia Virus (EIAV) is a lentivirus affecting horses, characterized by high genetic variability, complicating diagnosis.
  • Common diagnostic tests include:
    • Agar Gel Immunodiffusion Assay (AGID)
    • Enzyme-Linked Immunosorbent Assay (ELISA)
    • Western Blotting (WB)
  • Both AGID and ELISA traditionally detect antibodies primarily against the p26 capsid protein, mostly sourced from the Wyoming reference strain, which may limit detection of diverse viral variants.

Development of a Novel Multistrain ELISA

  • Researchers created a new indirect ELISA that targets antibodies against two viral proteins:
    • p26 (capsid protein)
    • gp45 (an envelope glycoprotein from two strains)
  • The purpose was to increase the antigenic diversity used in the assay to potentially improve detection across different EIAV strains.

Study Design and Methods

  • 372 serum samples were collected for analysis, including:
    • 96 samples positive for EIAV antibodies by all three ELISA tests
    • Other samples with varying statuses according to commercial tests
  • Three ELISA tests were evaluated:
    • The newly developed multistrain p26/gp45 indirect ELISA
    • Two existing commercial ELISAs commonly used in diagnostic labs
  • Statistical methods:
    • Cohen’s Kappa test: measures agreement between test results beyond chance
    • Bayesian Latent Class Analysis (BLCA): estimates diagnostic performance (sensitivity and specificity) without requiring a perfect reference test
  • Results were also compared with official classifications from the Italian Veterinary Service to assess real-world diagnostic performance.

Key Findings

  • Both Cohen’s Kappa and BLCA analyses demonstrated strong agreement and high diagnostic reliability for the two commercial ELISAs.
  • The novel p26/gp45 ELISA performed comparably, indicating it is an accurate and reliable diagnostic test.
  • Including the gp45 antigen from multiple EIAV strains appears to broaden detection capacity, potentially improving identification of diverse viral variants.
  • Comparison with official diagnostic classifications confirmed that the new ELISA aligns well with accepted standards.

Implications for Diagnostic Practice

  • This novel multistrain ELISA could serve as an enhancement or alternative to current commercial EIAV tests.
  • By increasing antigenic diversity in the assay, the test may reduce false negatives that arise due to viral genetic variability.
  • Its integration into routine diagnostic workflows could strengthen surveillance programs, especially in regions with diverse EIAV strains.
  • The study supports the application of Bayesian Latent Class Analysis as a valuable tool to evaluate diagnostic tests where no perfect reference standard exists.

Conclusion

  • The newly developed multistrain p26/gp45 indirect ELISA shows promise as a reliable and accurate diagnostic tool for detecting EIAV infection.
  • Its ability to recognize antibodies against multiple viral proteins and strains may help overcome limitations of current diagnostics driven by viral variability.
  • Wider adoption of this assay could improve EIAV diagnosis and control efforts worldwide.

Cite This Article

APA
Ostuni A, Frontoso R, Crudele MA, Barca L, Amati M, Boni R, De Vendel J, Raimondi P, Bavoso A. (2025). Comparative Evaluation of a Multistrain Indirect ELISA Targeting Anti- p26 and gp45 Antibodies for EIAV Detection. Pathogens, 14(6), 575. https://doi.org/10.3390/pathogens14060575

Publication

Pathogens (Basel, Switzerland)
ISSN: 2076-0817
NlmUniqueID: 101596317
Country: Switzerland
Language: English
Volume: 14
Issue: 6
PII: 575

Researcher Affiliations

Ostuni, Angela
  • Department of Basic and Applied Sciences, University of Basilicata, Via dell' Ateneo Lucano 10, 85100 Potenza, Italy.
Frontoso, Raffaele
  • Istituto Zooprofilattico Sperimentale del Mezzogiorno, Via Salute, 2, 80055 Portici, Italy.
Crudele, Maria Antonietta
  • Department of Basic and Applied Sciences, University of Basilicata, Via dell' Ateneo Lucano 10, 85100 Potenza, Italy.
Barca, Lorella
  • Istituto Zooprofilattico Sperimentale del Mezzogiorno, Via Salute, 2, 80055 Portici, Italy.
Amati, Mario
  • Department of Basic and Applied Sciences, University of Basilicata, Via dell' Ateneo Lucano 10, 85100 Potenza, Italy.
Boni, Raffaele
  • Department of Basic and Applied Sciences, University of Basilicata, Via dell' Ateneo Lucano 10, 85100 Potenza, Italy.
De Vendel, Jolanda
  • OneHEco APS, 84047 Capaccio Paestum, Italy.
Raimondi, Paolo
  • OneHEco APS, 84047 Capaccio Paestum, Italy.
Bavoso, Alfonso
  • Department of Basic and Applied Sciences, University of Basilicata, Via dell' Ateneo Lucano 10, 85100 Potenza, Italy.

MeSH Terms

  • Enzyme-Linked Immunosorbent Assay / methods
  • Antibodies, Viral / blood
  • Antibodies, Viral / immunology
  • Infectious Anemia Virus, Equine / immunology
  • Infectious Anemia Virus, Equine / isolation & purification
  • Animals
  • Horses
  • Equine Infectious Anemia / diagnosis
  • Equine Infectious Anemia / virology
  • Equine Infectious Anemia / immunology
  • Reproducibility of Results
  • Capsid Proteins / immunology
  • Sensitivity and Specificity

Conflict of Interest Statement

The authors declare no conflicts of interest.

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Citations

This article has been cited 1 times.
  1. Gonzálvez M, Franco JJ, Cano-Terriza D, Barbero-Moyano J, Jose-Cunilleras E, García J, Alguacil E, García-Bocanegra I. Equine Infectious Anemia Virus in Equids: A Large-Scale Serosurvey in Western Europe.. Animals (Basel) 2025 Dec 4;15(23).
    doi: 10.3390/ani15233499pubmed: 41375557google scholar: lookup
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