ELISA analysis of TNF-alpha was performed by coating 100 µL per well of anti-bovine TNF-alpha polyclonal antibody (Product # PBOTNFAI) at a concentration of 10 µg/mL and incubating overnight at 4C. The plate was blocked for one hour using 3% bovine serum albumin (BSA) diluted in PBST. The plate was washed six times with 200 µL/well PBST, and then incubated with 100 µL per well of recombinant bovine TNF-alpha protein standards and recombinant ovine TNF-alpha in tissue culture supernatant (both Log2 dilutions starting at an unknown concentration) for one hour at room temperature. The plate was washed with wash buffer six times and incubated with 100 µL per well anti-bovine TNF-alpha biotin-conjugated polyclonal antibody (Product # PBOTNFABI) at 1 µg/mL for one hour at room temperature. The plate was washed with wash buffer six times and then the signal was amplified using HRP-conjugated strepavidin at 2 µg/mL for one hour at room temperature. Detection was performed using 100 µL per well 3, 3', 5, 5'-tetramethylbenzidine (TMB) for 10 minutes. The reaction was stopped with 0.2M sulfuric acid. Absorbance in optical density (OD) values were read on a spectrophotometer at 450 nm. Data courtesy of the Innovators Program.