Sandwich ELISA analysis of Rat TNF-α matched pair antibodies Elisa assay was performed by coating wells of a 96-well plate with 100 µl per well of capture antibody (HA722170) diluted in carbonate/bicarbonate buffer, at a concentration of 2 µg/ml overnight at 4℃. Wells of the plate were washed, blocked with 150 µl 0.05% tween-20 1%BSA blocking buffer, and incubated with serial diluted Recombinant Rat TNF-α protein (HA210538) starting from 1000 pg/ml to 0 pg/ml and detect antibody (HA722171, Biotin, 0.2 µg/ml) for 1 hour at 30℃ with shaking. Then the plate was washed and incubated with 100 µl per well of SA-HRP for 0.5 hour at 30℃ with shaking. Detection was performed using an Ultra TMB Substrate for 10 minutes at room temperature in the dark. The reaction was stopped with sulfuric acid and absorbances were read on a spectrophotometer at 450 nm.