Human OX40/TNFRSF4 Recombinant Rabbit Monoclonal Antibody [PSH10-18] - BSA and Azide free (Detector)

Sandwich ELISA analysis of Human OX40/TNFRSF4 matched pair antibodies

Elisa assay was performed by coating wells of a 96-well plate with 50 µl per well of capture antibody (HA723187) 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 OX40/TNFRSF4 protein (HA210892) starting from 8,000 pg/ml to 0 pg/ml and detect antibody (HA723188, Biotin, 0.2 µg/ml) for 1 hour at 30℃ with shaking. Then the plate was washed and incubated with 50 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 OX40 in human PBMC cell culture supernatant.

PBMC cells were stimulated with 10 µg/ml PHA-M or vehicle control and incubated for 5 days. The concentrations of OX40 measured, interpolated from the OX40 standard curve and corrected for sample dilution. Undiluted samples are as follows: unstimulated 100% and stimulated 100%. The mean OX40 concentration was determined to be 418.6 pg/ml in PHA-M stimulated PBMC cell culture supernatant and undetectable in the unstimulated PBMC control.

Interpolated concentrations of spiked OX40 in human cell culture media samples.

The concentrations of OX40 were interpolated from the OX40 standard curves and corrected for sample dilution. Undiluted samples are as follows: cell culture media 1 50%, cell culture media 2 50%.