Goat anti rabbit igg conjugated with alkaline phosphatase

ELISA was used to identify Ab responses of individual rabbits to nTBI and TBI_tag. MaxiSorp 96-well plates (Thermo Fisher Scientific, USA) were coated with 5 μg/ml of nTBI and TBI_tag overnight at 4 °C in PBS. Plates were blocked for two hours with 0.5% casein in PBS at 37 °C and washed three times with 0.05% Tween 20/PBS (PBS-T). Eight serial dilutions of sera samples in PBS-0.1% casein (1/200, 1/1000, 1/5000 … 1/15,625,000) were added to the wells (100 μl per well) and incubated for two hours at 37 °C with shaking. Plates were washed three times with 0.05% PBS-T. Goat anti-rabbit IgG conjugated with alkaline phosphatase (Sigma, USA) were then added to the wells (100 μl/well) at 1:5000 dilution in PBS. Plates were incubated for one hour at room temperature and washed five times with 0.05% PBS-T. To visualize immunogen-specific antibodies BCIP/NBT substrate (Sigma, USA) was added to each well and incubated overnight in the dark at 37 °C. Plates were read at 405 nm (Model 680 Microplate reader, Bio-Rad, USA). Optical density (OD) values were calculated for each group by subtracting two times the average background of pre-immune rabbit serum.