Validation according to OIE criteria of a monoclonal, recombinant p26-based, serologic competitive enzyme-linked immunosorbent assay as screening method in surveillance programs for the detection of Equine infectious anemia virus antibodies.

The study presents the development and validation of a serologic test using a competitive enzyme-linked immunosorbent assay (cELISA), specifically designed for the detection of Equine infectious anemia (EIA) virus antibodies, developed by the Italian National Reference Center for EIA. The test demonstrated more effective detection rates of EIA cases than previously standard methods and fulfilled the World Organization for Animal Health standards.

Development and Validation of the Test

• The test developed by the Italian National Reference Center for EIA is a competitive enzyme-linked immunosorbent assay, or cELISA, that uses a recombinant p26-based monoclonal antibody to detect EIA virus antibodies in equine samples.
• The test was validated according to the standards set by the World Organization for Animal Health (OIE) in 2010. This involved evaluating several parameters of the test’s function and accuracy:
• Cutoff values, or the specific measurement above which a result is considered positive.
• Repeatability, which is the consistency of a test’s results under unchanged conditions.
• Reproducibility, which refers to the ability of a test to show consistent results when used under changed conditions.
• Concordance, or agreement between the results of the new test and a standard reference test.
• Analytical sensitivity, specificity, and diagnostic sensitivity and specificity all refer to the test’s ability to correctly identify positive and negative results. They collectively provide a measure of the test’s accuracy.
• The positive and negative predictive values of the test were also defined, relating to the test’s ability to correctly predict the presence or absence of disease relative to the actual disease status of the subjects.

Results of the Surveillance Program

• In comparison to the agar gel immunodiffusion test, the previously standard diagnostic method, the new cELISA test detected 17% more EIA cases when used to screen 96,468 samples in the Italian EIA surveillance program.
• The diagnostic specificity of the new cELISA test, as estimated from these samples, was 99.8%. This far exceeds the 80.2% diagnostic specificity rate that was estimated from the test’s initial validation process. Specificity refers to the test’s ability to correctly identify true negative results.
• The high sensitivity and specificity of the cELISA test, as concluded from these results, confirms its appropriateness for use as a screening test for equine infectious anemia. Sensitivity refers to the test’s ability to correctly identify true positive results.