Human TRAIL Recombinant Rabbit Monoclonal Antibody [PSH08-05] - BSA and Azide free (Capture)

Sandwich ELISA analysis of human TRAIL matched pair antibodies

Elisa assay was performed by coating wells of a 96-well plate with 100 µl per well of capture antibody (HA722943) diluted in carbonate/bicarbonate buffer, at a concentration of 5 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 Recombinant Human TRAIL protein as the standard (HA210893) starting from 2000 pg/ml to 0 pg/ml and detect antibody (HA722944, 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 TRAIL in 786-0 and Hela extract samples based on a 1000 µg/ml extract load.

Interpolated concentration of native TRAIL was measured in duplicate at different sample concentrations and interpolated from the TRAIL standard curves. The interpolated dilution factor corrected values were plotted (mean +/- SD, n=2). The mean TRAIL concentration was determined to be 11,821 pg/mL in 786-0 cell extract, There was no detectable signal in Hela cell extract.

Interpolated concentrations of native TRAIL in cell culture supernatant samples.

Interpolated concentration of native TRAIL was measured in duplicate at different sample concentrations and interpolated from the TRAIL standard curves. Undiluted samples were 100% cell supernatant. The interpolated dilution factor corrected values were plotted (mean +/- SD, n=2). The mean concentration was determined to be 260 pg/mL in 786-0 cell supernanant. There was no detectable signal in Hela cell supernatant.

Interpolated concentrations of spiked TRAIL in cell culture media samples.

The concentrations of TRAIL were measured in duplicates, interpolated from the TRAIL 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).