Igg elisa kits

Recombivirus^TM Mouse anti-ZIKV envelope protein IgG ELISA kits (Alpha Diagnostic International, RV-403120-1) were used according to the manufacturer’s protocol. In brief, 96-well plates coated with the ZIKV envelope protein were equilibrated with 300 μL of kit working wash buffer. Serial dilutions (3-fold) of sera from vaccinated mice were added. Diluted sera were incubated at RT for 1 h, and after four times washing buffer incubations, 100 μL/well of anti-mouse IgG HRP-conjugate working solution was added for 30 min at room temperature. Plates were washed 5 times and developed for 15 min at room temperature with 100 μL of (TMB) substrate (3,3′,5,5′-tetramethylbenzidine), then stopped by the addition of 100 μL of stop solution. Absorbance was measured at 450 nm on a microplate reader. ELISA ODs were compared between all vaccinated groups at different sera dilutions. For ELISA in BALB/c mice before challenge, antibody endpoint titres are the highest reciprocal serum dilution that resulted in an absorbance >2-fold over the background values, as calculate elsewhere [36 (link)]. Anti-Zika envelope antibody concentrations in A129 mice sera before challenge was measured by IgG ELISA using Zika Env antigen as previously described [48 (link)].

Sera obtained from dogs were tested to analyze the humoral responses to wt-RABV or TriGAS infection by ELISA. In order to determine the antibody responses against rabies infection, dog rabies virus and dog anti-rabies virus glycoprotein IgG ELISA kits from “Alpha Diagnostic” were used. Briefly, 100μl of diluted standards, controls and sera obtained from dogs were pipetted into the respective antigen coated 96 well plates and incubated at room temperature for 1 hour. Plates were then washed four times with wash buffer and 100μl of conjugate containing anti-dog IgG HRP was added. After 30 minutes incubation, plates were washed five times and 100μl of TMB substrate was added and incubated for 15 minutes. The color development was stopped by stop solution and plates were measured for OD at 450nm, using “BioTek” ELISA plate reader.