For binding ELISA, firstly MaxiSorp high-binding 96-well ELISA plates (ThermoFisher, USA)
were coated with different recombinant antigens at a concentration of 1μg/mL in PBS at 4°C
overnight. The plates were then washed 4 times with PBS containing 0.01% Tween-20 (PBST).
Subsequently, blocking was done with 10% FBS in PBS for 1 hour at 37°C. Serum samples
(collected from mice immunized with 50µg of PCaA-SEV at day 35) were serially diluted
(starting at 1:50, dilution factor 3.16) in PBS with 1% FBS. Then 100μl of the diluted
serum samples were added to the wells and incubated for 2 hours at 37°C. Following the
incubation, the plates were washed 4 times with PBST and incubated with HRP-conjugated
goat anti-mouse IgG (Sigma-Aldrich, St. Louis, MO, USA) for 1 hour at 37°C. Then 100μl of
3,3’5,5’-Tetramethylbenzidine (TMB) Substrate (Sigma-Aldrich, St. Louis, MO, USA) was
added to each well after the final wash and incubated for 10 minutes. The reaction was
stopped by addition of 100μl of 2N H2SO4 per well. Finally, the
optical density of the plate was measured at 450 nm using an ELISA plate reader (Biotek,
Winooski, VT, USA).
were coated with different recombinant antigens at a concentration of 1μg/mL in PBS at 4°C
overnight. The plates were then washed 4 times with PBS containing 0.01% Tween-20 (PBST).
Subsequently, blocking was done with 10% FBS in PBS for 1 hour at 37°C. Serum samples
(collected from mice immunized with 50µg of PCaA-SEV at day 35) were serially diluted
(starting at 1:50, dilution factor 3.16) in PBS with 1% FBS. Then 100μl of the diluted
serum samples were added to the wells and incubated for 2 hours at 37°C. Following the
incubation, the plates were washed 4 times with PBST and incubated with HRP-conjugated
goat anti-mouse IgG (Sigma-Aldrich, St. Louis, MO, USA) for 1 hour at 37°C. Then 100μl of
3,3’5,5’-Tetramethylbenzidine (TMB) Substrate (Sigma-Aldrich, St. Louis, MO, USA) was
added to each well after the final wash and incubated for 10 minutes. The reaction was
stopped by addition of 100μl of 2N H2SO4 per well. Finally, the
optical density of the plate was measured at 450 nm using an ELISA plate reader (Biotek,
Winooski, VT, USA).