Human CD28 Recombinant Rabbit Monoclonal Antibody [PSH12-50] - BSA and Azide free (Capture)

Sandwich ELISA analysis of Human CD28 matched pair antibodies

Capture: HA723457, Human CD28 Rabbit mAb [PSH12-50]
Detector: HA723458, Human CD28 Rabbit mAb [PSH12-51]

Elisa assay was performed by coating wells of a 96-well plate with 100 µl per well of capture antibody (HA723457) 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 CD28 protein (HA210920) starting from 10,000 pg/ml to 0 pg/ml and detect antibody (HA723458, 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 CD28 in human samples.

Capture: HA723457, Human CD28 Rabbit mAb [PSH12-50]
Detector: HA723458, Human CD28 Rabbit mAb [PSH12-51]

Interpolated concentration of native CD28 was measured in duplicate at different sample concentrations. Undiluted samples were 100% cell supernatant. The mean CD28 concentration was determined to be 275 pg/mL in Jurkat cell culture supernatant. There was no detectable signal in THP-1 cell culture supernatant.

Interpolated concentrations of native CD28 in Jurkat and THP-1 extract samples based on a 1000 µg/ml extract load.

Capture: HA723457, Human CD28 Rabbit mAb [PSH12-50]
Detector: HA723458, Human CD28 Rabbit mAb [PSH12-51]

Interpolated concentration of native CD28 was measured in duplicate at different sample concentrations and interpolated from the CD28 standard curves. The mean CD28 concentration was determined to be 4,345 pg/mL in Jurkat cell extract. There was no detectable signal in THP-1 cell extract.

Interpolated concentrations of spiked CD28 in cell culture media samples.

Capture: HA723457, Human CD28 Rabbit mAb [PSH12-50]
Detector: HA723458, Human CD28 Rabbit mAb [PSH12-51]

The concentrations of CD28 were measured in duplicates, interpolated from the CD28 standard curves and corrected for sample dilution. Undiluted samples are as follows: cell culture media 50%.