Development of IgM-ELISA for diagnosis of recent infection of Japanese encephalitis virus in equines.

The research aimed to establish a highly sensitive diagnostic tool, specifically IgM-ELISA, for early detection of the Japanese encephalitis virus (JEV) in horses, a prevalent mosquito-borne disease with a high infection rate in Indian equines.

Development of IgM-ELISA Diagnostic Tool for JEV

• The researchers focused on detecting specific immunoglobulin ‘M’ (IgM) in horses, since it serves as a signifier of recent JEV infection. They aimed to build a sensitive and specific recombinant NS1 protein-based indirect IgM-ELISA and IgM capture (MAC) ELISA diagnostic tools.
• The indirect IgM ELISA they developed showed excellent diagnostic sensitivity and specificity of 100% and 88.5%, respectively. Various laboratories validated its reproducibility with a high Cohen’s kappa value range of 0.84 to 1. This suggests that the test provides consistent, reliable results across different settings.

Challenges in Standardizing MAC ELISA

• While trying to standardize MAC ELISA using the checker board titration method, the researchers observed unwanted binding of polyclonal anti-equine IgM capture antibody with anti-porcine IgG conjugate and with hyperimmune serum made in swine against the antigen. This highlighted the need to use monoclonal capture antibody.
• However, the performance of MAC ELISA, even when standardized with the monoclonal antibody, did not meet satisfactory limits, indicating its potential ineffectiveness as a diagnostic tool for JEV in horses.

Screening and Implications for Equine Healthcare

• The researchers employed the more efficient IgM-ELISA to screen 821 equine serum samples, out of which they discovered a 33.73% positivity rate of IgM antibodies against JEV in Indian horse populations.
• The high seropositivity to JEV underscores the necessity for vaccinations, as well as research focused on antiviral therapy for the equine population in India.
• The successful development of indirect IgM-ELISA provides a vital tool not only for diagnosing acute or recent infection of JEV in horses, but also in conducting sero-epidemiological studies on the spread and control of the disease.