Complement-Dependent Cytotoxicity Assay for Differentiating West Nile Virus from Japanese Encephalitis Virus Infections in Horses

Abstract

A complement-dependent cytotoxicity (CDC) assay was established to measure antibodies to the West Nile virus (WNV) nonstructural protein 1 (NS1) in horses. Sera collected from a WNV-infected horse mediated lysis of WNV NS1-expressing cells in a dose-dependent manner at higher percentages than sera from a Japanese encephalitis virus (JEV)-infected horse. The percentages of specific lysis for sera diluted 1:10 to 1:80 were <19.8% (assay cutoff) for almost all of the 100 JEV-infected or uninfected horses tested, in contrast to 55 to 76% in WNV-infected horses. Experimental infection revealed that horses became anti-WNV NS1 antibody positive 10 days after WNV infection. This study demonstrated the utility of this assay for differentiating WNV from JEV infections in horses.

Nonstructural Protein 1-Specific Immunoglobulin M and G Antibody Capture Enzyme-Linked Immunosorbent Assays in Diagnosis of Flaviviral Infections in Humans

“…b Control panels were other serocomplex panels and negative panels. lished indirect ELISA, an epitope-blocking ELISA, or a complement-dependent cytotoxicity assay (29)(30)(31)(32), the use of an NS1-MAC-ELISA has not been reported, and validated test kits…”

Specific Antibody Responses to West Nile Virus Infections in Horses Preimmunized with Inactivated Japanese Encephalitis Vaccine: Evaluation of Blocking Enzyme-Linked Immunosorbent Assay and Complement-Dependent Cytotoxicity Assay

“…The CDC assay was performed as previously described (Kitai et al 2010). Briefly, 50 mL of heat-inactivated test serum was mixed with an equal volume of 2G2 cell suspension containing 5Â10 4 cells and incubated on ice for 30 min.…”

“…Both assay methods successfully detected antibodies reactive to WNV but not to JEV. However, horses used for these experiments (Kitai et al 2007(Kitai et al , 2010 were naïve, representing primary infection with WNV but not adapting to the situation in Japan where almost all horses are immune to JEV.…”

“…The blocking ELISA is based on the binding of a specific antibody to the antigen that is inhibited by its preceding incubation with a test antibody specimen (Kitai et al 2007). The CDC assay utilizes a mechanism of antibody-mediated CDC in which complement activation triggered by a specific antigenantibody binding on the cell surface causes cell lysis (Kitai et al 2010). For these assays, we used the nonstructural protein 1 (NS1) of WNV as an antigen: We generated a monoclonal antibody specific for the NS1 of WNV and also a cell line stably expressing the NS1 antigen of WNV on the cell surface and in a secreted form (Kitai et al 2007).…”

“…Our laboratory has developed an epitope-blocking ELISA and a complement-dependent cytotoxicity (CDC) assay for detecting antibody responses specific for WNV, but not crossreactive to JEV in horse sera (Kitai et al 2007(Kitai et al , 2010. The blocking ELISA is based on the binding of a specific antibody to the antigen that is inhibited by its preceding incubation with a test antibody specimen (Kitai et al 2007).…”