Human sTNF RI Recombinant Rabbit Monoclonal Antibody [PSH09-89] - BSA and Azide free (Capture)

Sandwich ELISA analysis of Human sTNF RI matched pair antibodies

Elisa assay was performed by coating wells of a 96-well plate with 100 µl per well of capture antibody (HA723154) diluted in carbonate/bicarbonate buffer, at a concentration of 2ug/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 Human sTNF RI Protein (HA210562) starting from 10000 pg/ml to 0 pg/ml and detect antibody (HA723159, 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.

Interpolated concentrations of native sTNF R1 in human samples.

Interpolated concentration of native sTNF R1 was measured in duplicate at different sample concentrations and interpolated from the sTNF R1 standard curves. The interpolated dilution factor corrected values were plotted (mean +/- SD, n=2). The mean sTNF R1 concentration was determined to be 3,727 pg/mL in human urine and 1,145 pg/ml in A549 cell supernatant. There was no detectable signal in Raji supernatant.

Interpolated concentrations of spiked sTNF RI in cell culture media samples.

The concentrations of sTNF RI were measured in duplicates, interpolated from the sTNF RI standard curves and corrected for sample dilution. Undiluted samples are as follows: cell culture media 50%. The interpolated dilution factor corrected values are plotted (mean +/- SD, n=2).