Standard curve of TNF-alpha matched pair antibodies: Sandwich ELISA analysis of TNF-alpha matched pair antibodies Elisa assay was performed by coating wells of a 96-well plate with 50 µl per well of capture antibody HA721265 [PS00-40] 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 1% BSA/PBST blocking buffer, and incubated with serial diluted recombinant TNF-alpha protein starting from 2000 pg/ml to 31.25 pg/ml for 1 hour at 37℃. The plate was washed and incubated with 50 µl per well of detect antibody [PS01-07] (Biotin, 1:4,000) for 1 hour at 37℃. Then the plate was washed and incubated with 50 µl per well of Streptavidin-HRP for 0.5 hour at 37℃. 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. Capture/detect antibodies, recombinant TNF-alpha protein, Streptavidin-HRP are diluted in 1% BSA/PBST.