Peroxidase affinipure goat anti syrian hamster igg h l

Nunc MaxiSorp flat- bottom 96 well plates were coated with recombinant SARS-CoV-2 RBD- His recombinant protein (40595-V80H, Sino Biological, China) and incubated overnight at 4 °C.
After the incubation, plates were washed with PBS containing 0.1% Tween-20 plates were blocked with 3% Bovine Serum Albumin (IgG-Free). Hamster serum was diluted (fivefold serial dilution) from 1:100 up to 1:1562500. Diluted serums were added to the plate and incubated for 1 h at 37 °C. Next, plates were washed with PBS-T and Peroxidase AffiniPure Goat Anti-Syrian Hamster IgG (H + L) (Cat # 107-035-142, Jackson Immuno Research, West Grove, USA) was added to each well and incubated at 37 °C for 1 h. After the last wash with PBS-T, 100 µL of Tetramethylbenzidine (TMB) substrate (Cat# 7004P6, Cell Signaling Technology, MA, USA) was added to each well. After a two-minute incubation at room temperature, 100 µL of Stop solution (Cat# 7002P6, Cell Signaling Technology, MA, USA) was added to terminate the reaction and absorbance was measured at 450 nm. Inhibitory dilution 50 (ID50) was calculated using non-linear regression analysis.

Nunc MaxiSorp™ flat-bottom 96-well plates (ThermoFisher, Ottawa, ON) were coated with 1 µg/mL of either SARS-CoV-2 Spike S1+S2 ECD-His recombinant protein or SARS-CoV-2 Spike RBD-His recombinant protein (Sino Biological, Beijing, China) in PBS and incubated overnight at 4°C. Plates were washed with PBS containing 0.1% Tween-20 (PBS-T) before blocking with 3% (w/v) Bovine Serum Albumin (IgG-Free, Protease-Free) (Jackson Immuno Research, West Grove, PA) in PBS-T for 2 hours at 37°C. The plates were washed again and two-fold serial dilutions of hamster serum, starting from 1:50 up to 1:102400 were added to the wells and incubated for 1 hour at 37°C. Plates were then washed with PBS-T and Peroxidase AffiniPure Goat Anti-Syrian Hamster IgG (H+L) (Jackson Immuno Research, West Grove, PA) was added to each well at 1:4000 and incubated at 37°C for 1h. Plates were washed again with PBS-T and 100 µL of Tetramethylbenzidine (TMB) substrate (Cell Signaling Technology, Danvers, MA) was added to each well. After a two-minute incubation at room temperature, 100 µL of 0.16 M sulfuric acid was added to terminate the reaction and absorbance was measured at 450 nm. Endpoint titers were expressed as the reciprocals of the final detectable dilution with an OD above the cut-off value, which was defined as the average OD of the pcDNA3.1-empty samples plus 3 standard deviations.