Mouse/Rat TNF alpha Recombinant Rabbit Monoclonal Antibody [PSH03-29] - BSA and Azide free (Capture)

Sandwich ELISA analysis of rat TNF alpha matched pair antibodies

Elisa assay was performed by coating wells of a 96-well plate with 100 µl per well of capture antibody (HA721974) 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 rat TNF alpha protein (HA210538) starting from 3000 pg/ml to 0 pg/ml and detect antibody (HA721975) -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.

Sandwich ELISA analysis of mouse TNF alpha matched pair antibodies

Elisa assay was performed by coating wells of a 96-well plate with 100 µl per well of capture antibody (HA721974) 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 mouse TNF alpha protein (HA210527) starting from 3000 pg/ml to 0 pg/ml and detect antibody (HA721975)-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.

Conditioned media was harvested after 72 hours. TNF-α was measured in 100% unstimulated and LPS stimulated rat spleen cell supernatant. The concentrations of TNF-α were interpolated from the TNF-α standard curves. The mean TNF-α concentration was determined to be 128 pg/mL in LPS stimulated rat spleen cell supernatant. There was no detectable signal in unstimulated supernatant.