Enhanced sensitivity of an antibody competitive blocking enzyme-linked immunosorbent assay using Equine arteritis virus purified by anion-exchange membrane chromatography.

The research aimed to enhance a method for detecting antibodies to Equine arteritis virus (EAV). The researchers used anion-exchange membrane chromatography to purify the EAV, which they found increased the effectiveness of the competitive blocking enzyme-linked immunosorbent assay (cELISA) test.

Research Methodology and Assessment

• The objective was to improve an existing cELISA test used for detecting antibodies to EAV by purifying the virus antigen using anion-exchange membrane chromatography capsule (AEC).
• The AEC method proved to be rapid and easily scalable for virus purification.
• Researchers compared the effectiveness of the virus purified by the AEC method with that acquired by differential centrifugation. The two methods were compared based on the quality and purity of EAV glycoprotein 5 (GP5), which contains the epitope defined by monoclonal antibody 17B7, and the sensitivity of a commercial antibody cELISA implementing the two different purified antigens.

Anion-exchange membrane chromatography results

• The AEC-purified EAV showed a high relative amount of GP5 monomer, with 86%, while the EAV obtained through differential centrifugation contained less than 29% of the monomer. The GP5 monomer was evaluated by Western blot testing using GP5-specific monoclonal antibody 17B7.
• Significant improvements in analytical sensitivity without compromising analytical specificity were observed when cELISAs with both purification methods were evaluated using seven sensitivity and specificity check sets.
• Further, the cELISA using AEC-purified EAV showed 30-40% higher agreement with the virus neutralization (VN) test in comparison with the cELISA based on differentially centrifuged EAV, as tested on 40 borderline EAV-seropositive samples.
• Also, the AEC-based cELISA demonstrated superior robustness, with a significant statistical variance (P = 0.001), evident by its consistency within the same laboratory (intra-laboratory repeatability) and between different laboratories (interlaboratory reproducibility).

Conclusion and Implications

• The researchers concluded that using AEC-purified EAV in the cELISA should enhance the harmonization of the cELISA with the World Organization for Animal Health-prescribed VN test.
• This improvement is expected to lead to a more accurate diagnosis and better management of EAV, which is critical for the health and wellbeing of equine populations.