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Equine veterinary journal2022; 55(1); 111-121; doi: 10.1111/evj.13555

Validation of an indirect in-house ELISA using synthetic peptides to detect antibodies anti-gp90 and gp45 of the equine infectious anaemia virus.

Abstract: Equine infectious anaemia (EIA) is controlled by the identification of seropositive animals. The official diagnostic method is the agar gel immunodiffusion (AGID) test, which detects antibodies against a viral core protein (p26). Although AGID is inexpensive and specific, the report of results takes considerable time and the test has low analytical sensitivity. Objective: To validate our in-house indirect ELISA , following the World Organization of Animal Health (OIE) criteria. Methods: Test validation. Methods: Synthetic peptides gp90 and gp45 were used as antigens in ELISA . Tests used for validation, calibration and linear working operating range, analytical and diagnostic sensitivity and specificity, repeatability and reproducibility were assessed by comparing them with the AGID test and using 1844 equine sera grouped into five different panels. Results: We were able to replace the National References Sera with our Internal Reference Sera. ELISA had acceptable repeatability and reproducibility. Analytical sensitivity of the ELISA was 800 times greater than that of AGID test for positive sera and 400 times greater for weak positive sera. ELISA also showed optimal analytical specificity, since no cross-reactivity was detected with antibodies against other equine viruses. One sample was positive by AGID test and negative by ELISAgp90/45. ELISA was performed using 243 EIA positive and 878 negative equid sera, and showed a diagnostic sensitivity of 99.59% [CI 97.73%-99.99%] and a diagnostic specificity of 90.32% [CI 88.17%-92.19%], compared to AGID test; thus, it was demonstrated to be a robust test. Conclusions: Samples were derived from naturally infected equid populations showing heterogeneous clinical states: therefore, their status was uncertain and some horses were sampled more than once. The AGID test may not be the most useful gold standard. Conclusions: ELISA is a useful tool for the diagnosis of EIAV infection and meets validation requirements established by the OIE.
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Publication Date: 2022-03-01 PubMed ID: 35007356DOI: 10.1111/evj.13555Google 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.

The research focuses on the development and validation of a more efficient method, known as indirect ELISA, for detecting equine infectious anaemia (EIA) in horses. EIA is currently diagnosed through the agar gel immunodiffusion (AGID) test but this method has limitations, specifically lower sensitivity and longer result reporting time.

Methodology

  • The researchers developed an in-house indirect Enzyme-Linked Immunosorbent Assay (ELISA) using synthetic peptides gp90 and gp45 as antigens.
  • To validate the ELISA test, researchers used 1844 equine sera samples that were grouped into five different panels.
  • Multiple test criteria were evaluated such as validation, calibration, linear working operating range, analytical and diagnostic sensitivity and specificity, and repeatability and reproducibility.
  • The results obtained from the ELISA tests were then compared with the results from the AGID tests.

Results

  • The researchers found satisfactory repeatability and reproducibility in the ELISA method.
  • The analytical sensitivity of the ELISA test was found to be significantly higher compared to the AGID test; 800 times greater for positive sera and 400 times greater for weak positive sera.
  • No cross-reactivity was noted with antibodies against other equine viruses, implying excellent analytical specificity of the ELISA test.
  • One sample was found to be positive in AGID but negative in the ELISA test, indicating some variability in the tests.
  • When used on 243 EIA positive and 878 negative equid sera, ELISA showed a diagnostic sensitivity of 99.59% and a diagnostic specificity of 90.32%, compared to the AGID test.

Conclusions

  • The researchers highlighted variability in the clinical status of the equine populations tested due to natural infection, which may have resulted in uncertain statuses and multiple samplings from some horses.
  • Despite this, the researchers concluded that ELISA represents a significant improvement over the traditional AGID test for diagnosing EIA, as it offers higher sensitivity, faster results, and minimal cross-reactivity with antibodies for other equine viruses.
  • It meets the validation requirements set by the World Organization of Animal Health (OIE) and is thus a viable alternative for diagnosing EIA.

Cite This Article

APA
Russi RC, Garcia L, Cámara MS, Soutullo AR. (2022). Validation of an indirect in-house ELISA using synthetic peptides to detect antibodies anti-gp90 and gp45 of the equine infectious anaemia virus. Equine Vet J, 55(1), 111-121. https://doi.org/10.1111/evj.13555

Publication

Equine veterinary journal
ISSN: 2042-3306
NlmUniqueID: 0173320
Country: United States
Language: English
Volume: 55
Issue: 1
Pages: 111-121

Researcher Affiliations

Russi, Romina Cecilia
  • Laboratorio de Diagnóstico e Investigaciones Agropecuarias., Ministerio de la Producción, Ciencia y Tecnología de la Provincia de Santa Fe, Santa Fe, Argentina.
  • Laboratorio de Inmunología Experimental, Cátedra de Inmunología Básica, Facultad de Bioquímica y Ciencias Biológicas, Universidad Nacional del Litoral, Santa Fe, Argentina.
Garcia, Lucila
  • Laboratorio de Diagnóstico e Investigaciones Agropecuarias., Ministerio de la Producción, Ciencia y Tecnología de la Provincia de Santa Fe, Santa Fe, Argentina.
Cámara, María Silvia
  • Laboratorio de Control de Calidad de Medicamentos, Cátedra de Control de Calidad, Facultad de Bioquímica y Ciencias Biológicas, Universidad Nacional del Litoral, Santa Fe, Argentina.
Soutullo, Adriana Rosa
  • Laboratorio de Diagnóstico e Investigaciones Agropecuarias., Ministerio de la Producción, Ciencia y Tecnología de la Provincia de Santa Fe, Santa Fe, Argentina.
  • Laboratorio de Inmunología Experimental, Cátedra de Inmunología Básica, Facultad de Bioquímica y Ciencias Biológicas, Universidad Nacional del Litoral, Santa Fe, Argentina.

MeSH Terms

  • Horses
  • Animals
  • Infectious Anemia Virus, Equine
  • Reproducibility of Results
  • Equine Infectious Anemia / diagnosis
  • Enzyme-Linked Immunosorbent Assay / veterinary
  • Antibodies, Viral
  • Peptides
  • Immunodiffusion / veterinary
  • Horse Diseases

Grant Funding

  • Ministerio de la Producciu00f3n, Ciencia y Tecnologu00eda de la Provincia de Santa Fe
  • Facultad de Bioquu00edmica y Ciencias Biolu00f3gicas de la Universidad de Santa Fe
  • Fundaciu00f3n Nuevo Banco de Santa Fe

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Citations

This article has been cited 5 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
  2. Firdausy LW, Fikri F, Wicaksono AP, Çalışkan H, Purnama MTE. Global prevalence and risk factors of equine infectious anemia: A systematic review and meta-analysis. Vet World 2025 Jun;18(6):1440-1451.
    doi: 10.14202/vetworld.2025.1440-1451pubmed: 40689174google scholar: lookup
  3. Wang J, Qiu J, Wang M, Wu X, Li X, Zhang H. Development of a colloidal gold immunochromatographic strip to detect equine infectious anemia virus. Virol J 2025 Jun 24;22(1):205.
    doi: 10.1186/s12985-025-02815-6pubmed: 40555999google scholar: lookup
  4. Domínguez-Odio A, González LIC, Alfonso DM, Guevara-Hernández F, La O Arias MA, Zayas MP, Ríos MÁB. Serodiagnosis of equine infectious anemia by indirect ELISA based on a novel synthetic peptide derived from gp45 glycoprotein. Vet Res Commun 2025 Apr 22;49(3):174.
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  5. Zhang Z, Guo K, Chu X, Liu M, Du C, Hu Z, Wang X. Development and evaluation of a test strip for the rapid detection of antibody against equine infectious anemia virus. Appl Microbiol Biotechnol 2024 Dec;108(1):85.
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