Direct ELISA analysis of TNF alpha was performed by coating wells of a 96-well plate with 100ul per well of human recombinant protein TNF alpha diluted in carbonate/bicarbonate buffer at a concentration of 1 µg/mL overnight at 4C. Wells of the plate were washed, blocked with starting blocking buffer, and incubated with 100ul per well of a rabbit anti-TNF alpha polyclonal antibody at a dilution of 1:25, 1:50, 1:100, 1:200, 1:400, 1:800, 1:1600 and 1:3200 for 90 minutes at 37C. The plate was washed, then incubated with 100ul per well of a HRP-conjugated goat anti-rabbit IgG secondary antibody at a dilution of 1:2000 for 90 minutes at 37C. Detection was performed using 1-Step Ultra TMB substrate for 5-10 minutes at room temperature in the dark. The reaction was stopped with Stop solution, and absorbances were read on a spectrophotometer at 450-550nm.