Groups of six mice were immunized at 3-week intervals (on Weeks 0, 3 and 6) and blood samples collected
on Weeks 5 and 8. ELISAs to measure the titers Belnacasan supplier of OVA-specific IgG subtypes were performed similarly, with minor modifications. In this instance, the initial dilutions of serum samples were 1:3000 and 1:100 for the IgG1 and IgG2a antibody-binding assays, respectively, and in the next step, the secondary antibodies (goat anti-mouse IgG1 and IgG2a [Southern Biotech, Birmingham, AL, USA]; 1:4000) were assessed. Figure 5a shows that at Week 5 there were no significant differences in OVA-specific IgG1 or IgG2a titers compared with controls among mice immunized with pyriproxyfen and alum. Figure 5b shows that pyriproxyfen significantly enhanced OVA-specific IgG2a titers compared to controls at 8 weeks (eightfold greater; P = 0.002), whereas the difference find more in the OVA-specific IgG1 immune response compared to the control remained insignificant. As expected, immunization with OVA containing alum resulted in a significantly greater OVA-specific IgG1 titer (fourfold greater, P = 0.01) than in the control at 8 weeks (Fig. 5b). These observations suggest that the IgG subtypes assessed, IgG2a and IgG1, reached significantly increased titers after immunization three times with pyriproxyfen
or alum in OVA. The titers of IgE were also measured to determine the effect of pyriproxyfen on IgE production. For this, mice were immunized three times with OVA (in 5% ethanol) alone or with pyriproxyfen (15 mM) or alum and the titers 17-DMAG (Alvespimycin) HCl of IgE measured. Groups of six mice were immunized at 3 week intervals (Weeks 0, 3 and 6) and blood samples collected on Weeks 8. ELISA for measuring the IgE titer was performed according to a method similar to that
described above except the initial dilution of serum samples was 1:10 for the IgE antibody binding assay and the secondary antibody used was goat anti-mouse IgE (Southern Biotech) (1:4000). As shown in Figure 5c, there were no significant differences in OVA-specific IgE titer between mice immunized with OVA plus pyriproxyfen and controls. Compared to the controls, at 8 weeks OVA-specific IgE titers were increased only in mice immunized with OVA containing alum (P = 0.01). Cytokine profiles were also checked to confirm the basis for immune responses after the addition of pyriproxyfen. Two groups of five mice were immunized on Weeks 0, 3 and 6 and injected with OVA (in 5% ethanol) with or without pyriproxyfen (15 mM) and alum, prior to spleen collection on Weeks 3 and 8 and measurement of cytokine concentrations by sandwich ELISA. The spleens were dissected out from the mice under aseptic conditions. Single-cell suspensions were prepared by homogenizing each spleen in 3 mL of RPMI 1640 medium (Sigma–Aldrich) followed by centrifugation for 5 mins at 1200 rpm at 4°C.