Streptavidin conjugated horseradish peroxidase sa hrp and tmb substrate

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.

The functional analysis of CD4⁺ T cell responses has been published previously [66 (link)]. Briefly, CD4⁺ T cells were MACS-purified from the draining lymph nodes with anti-CD4 (Miltenyi Biotec, Auburn, CA). Subsequently, isolated CD4⁺ T cells were used to directly measure cytokine gene expression by qPCR. Alternatively, 2 x 10⁵ CD4⁺ T cells/well were cultured in 96-well U-bottom plates in the presence of 5μg E651 peptide and 3 x 10⁵ naïve splenocytes as antigen-presenting cells. IL-21 production in the cultures was measured three days later by either qPCR. IFN-γ protein was measured by ELISA with an anti-mouse IFN-γ capture antibody, followed by an biotinylated anti-mouse IFN-γ detection antibody, and subsequently streptavidin-conjugated horseradish peroxidase (SA-HRP) and TMB substrate (both BD Bioscience, San Diego, CA).