Standardization and validation of an agar gel immunodiffusion test for the diagnosis of equine infectious anemia using a recombinant p26 antigen.
Abstract: We developed and validated an agar gel immunodiffusion test (AGID) test for the diagnosis of equine infectious anemia (EIA) using as antigen the p26 protein of equine infectious anemia virus (EIAV) produced in the Escherichia coli expression system. The developed rp26-AGID test showed an excellent diagnostic relative sensitivity (100%) and specificity (100%) compared to a commercial AGID assay when 1855 field serum samples were analyzed. In addition, the rp26-AGID demonstrated to be a precise assay with excellent repeatability and reproducibility. In the analytical sensitivity trial, positive sera showed nearly the same endpoint dilutions for both compared tests. No positive-reactions were observed with 35 serum samples with antibodies related to other endemic agents and also with severely hemolysed samples, demonstrating that the rp26-AGID has an excellent analytical specificity. Complete concordance with blind previous results from five proficiency test panels confirmed the capability of the assay of accurate detection of EIAV antibodies. This is the first time that a recombinant AGID assay able to identify EIAV infections has been standardized and validated in Argentina according to international guidelines. Taking into account the results obtained, the p26-AGID could be adopted as an official test method for the diagnosis and control of EIA in this country.
Publication Date: 2007-01-17 PubMed ID: 17292568DOI: 10.1016/j.vetmic.2007.01.007Google Scholar: Lookup
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- Comparative Study
- Journal Article
- Research Support
- Non-U.S. Gov't
- Validation Study
- Antibodies
- Antigen
- Clinical Study
- Diagnosis
- Diagnostic Technique
- Disease control
- Disease Diagnosis
- Disease Treatment
- Epidemiology
- Equine Diseases
- Equine Health
- Equine Infectious Anemia
- Escherichia coli
- Horses
- Immunology
- Infection
- Infectious Disease
- Laboratory Methods
- Serodiagnosis
- Veterinary Medicine
- Veterinary Research
Summary
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This research aimed to develop and validate an agar gel immunodiffusion test using a recombinant p26 antigen for the diagnosis of equine infectious anemia. The results showed this test to be highly effective, producing consistent and accurate results. It is suggested that this testing method could be officially adopted for the diagnosis and control of equine infectious anemia in Argentina.
Development and Validation of the Test
- A new agar gel immunodiffusion test (AGID) was developed for the diagnosis of equine infectious anemia (EIA).
- The test uses the p26 protein obtained from the equine infectious anemia virus (EIAV) as an antigen. This protein was produced in an Escherichia coli expression system.
- The developed test, referred to as the rp26-AGID, was validated and showed excellent diagnostic relative sensitivity and specificity. It was accurate in identifying of EIA in horses.
Testing Methodology and Results
- The validation of rp26-AGID was conducted using 1855 field serum samples. The performance of rp26-AGID was compared to a commercial AGID test, showing a 100% match in sensitivity and specificity.
- In addition to its accuracy, the rp26-AGID displayed excellent repeatability and reproducibility, demonstrating the precision of the test.
- An analytical sensitivity trial was conducted during which positive sera displayed almost identical endpoint dilutions for both compared tests.
Analytical Specificity and Compatibility
- An important feature of rp26-AGID is its analytical specificity. No positive reactions were observed when the test was conducted with 35 serum samples that had antibodies related to other endemic agents.
- Also, no false positives were identified when severely hemolysed samples were tested.
- A 100% match was observed with blind previous results, verifying the accuracy of rp26-AGID in the detection of EIAV antibodies.
Conclusion and Recommendations
- This is reportedly the first time that a recombinant AGID test for EIA has been standardized and validated in Argentina as per international guidelines.
- The rp26-AGID, given its perfect sensitivity and specificity, is recommended as an official testing method for the diagnosis and control of EIA in Argentina.
Cite This Article
APA
Alvarez I, Gutierrez G, Vissani A, Rodriguez S, Barrandeguy M, Trono K.
(2007).
Standardization and validation of an agar gel immunodiffusion test for the diagnosis of equine infectious anemia using a recombinant p26 antigen.
Vet Microbiol, 121(3-4), 344-351.
https://doi.org/10.1016/j.vetmic.2007.01.007 Publication
Researcher Affiliations
- Instituto de Virología, Centro de Investigaciones en Ciencias Veterinarias y Agronómicas, INTA, C.C. 1712 Castelar, Argentina. ialvarez@cnia.inta.gov.ar
MeSH Terms
- Animals
- Base Sequence
- Equine Infectious Anemia / diagnosis
- Equine Infectious Anemia / virology
- Immunodiffusion / methods
- Immunodiffusion / standards
- Immunodiffusion / veterinary
- Infectious Anemia Virus, Equine / genetics
- Infectious Anemia Virus, Equine / immunology
- Infectious Anemia Virus, Equine / isolation & purification
- Molecular Sequence Data
- RNA, Viral / chemistry
- RNA, Viral / genetics
- Recombinant Proteins / chemistry
- Recombinant Proteins / genetics
- Recombinant Proteins / immunology
- Reproducibility of Results
- Reverse Transcriptase Polymerase Chain Reaction / veterinary
- Sensitivity and Specificity
- Viral Core Proteins / chemistry
- Viral Core Proteins / genetics
- Viral Core Proteins / immunology
Citations
This article has been cited 8 times.- Hu Z, Guo K, Du C, Sun J, Naletoski I, Chu X, Lin Y, Wang X, Barrandeguy M, Samuel M, Wang W, Lau PI, Wernery U, Raghavan R, Wang X. Development and evaluation of a blocking ELISA for serological diagnosis of equine infectious anemia. Appl Microbiol Biotechnol 2023 May;107(10):3305-3317.
- Kwit E, Osiński Z, Lavazza A, Rzeżutka A. Detection of Myxoma Virus in the Classical Form of Myxomatosis Using an AGID Assay: Statistical Assessment of the Assay's Diagnostic Performance. J Vet Res 2020 Sep;64(3):369-372.
- Liu Q, Ma J, Wang XF, Xiao F, Li LJ, Zhang JE, Lin YZ, Du C, He XJ, Wang X, Zhou JH. Infection with equine infectious anemia virus vaccine strain EIAVDLV121 causes no visible histopathological lesions in target organs in association with restricted viral replication and unique cytokine response. Vet Immunol Immunopathol 2016 Feb;170:30-40.
- Alvarez I, Gutierrez G, Ostlund E, Barrandeguy M, Trono K. Western blot assay using recombinant p26 antigen for detection of equine infectious anemia virus-specific antibodies. Clin Vaccine Immunol 2007 Dec;14(12):1646-8.
- Ostuni A, Frontoso R, Crudele MA, Barca L, Amati M, Boni R, De Vendel J, Raimondi P, Bavoso A. Comparative Evaluation of a Multistrain Indirect ELISA Targeting Anti- p26 and gp45 Antibodies for EIAV Detection. Pathogens 2025 Jun 8;14(6).
- 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.
- Ma X, Gao M, Zhang X, Ma W, Xue F, Wang XF, Wang X. Identification and characterization of linear epitopes of monoclonal antibodies against the capsid proteins of small ruminant lentiviruses. Front Microbiol 2024;15:1452063.
- 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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