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

BACKGROUND

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.

STUDY DESIGN

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.

MAIN LIMITATIONS

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.

CONCLUSION

ELISA is a useful tool for the diagnosis of EIAV infection and meets validation requirements established by the OIE.