Rapid ELISA for WNV Antibodies

Detection of WNV-specific antibodies was based on ELISAs with recombinant WNV-E, DIII, or DIII-KT as antigens. Production of these reagents followed published protocols [77 (link),78 (link)]. Briefly, DIII proteins were expressed in BL21(DE3) E. coli cells, refolded from inclusion bodies by oxidative refolding, and purified by size exclusion. AviTag-DIII protein was biotinylated and purified again by size exclusion. Serial dilutions of serum from RWN-infected mice were applied to plates coated with recombinant WNV-E, DIII; or DIII-KT proteins. Bound RWN-specific antibodies were detected with biotinylated goat anti-mouse IgM, IgG, IgG2b, IgG2c, or IgG3 (Southern Biotech, Birmingham, AL), followed by streptavidin-conjugated horseradish peroxidase (SA-HRP) and TMB substrate (both BD Bioscience, San Diego, CA). Anti-mouse Ig(H+L) (Southern Biotech, Birmingham, AL) and serial dilutions of mouse IgM and IgG2c (Southern Biotech, Birmingham, AL) were used for standards. High-affinity antibodies were measured similarly using plates coated with recombinant DIII alone or diluted 1:3 with BSA. After the initial binding, low affinity antibodies were washed off by incubating the samples for 15 min in presence of increasing amounts of NaSCN before detection with biotinylated goat anti-mouse IgM or IgG, followed by SA-HRP.