Human GITR Recombinant Rabbit Monoclonal Antibody [PSH05-63] - BSA and Azide free (Capture)

Sandwich ELISA analysis of human GITR matched pair antibodies

Elisa assay was performed by coating wells of a 96-well plate with 100 µl per well of capture antibody (HA722383) 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 0.05% tween-20 1%BSA blocking buffer, and incubated with serial diluted recombinant human GITR protein (HA210945) starting from 3000 pg/ml to 0 pg/ml and detect antibody (HA722384, 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 GITR in Jurkat and HuT-102 cell extract samples based on a 1000 µg/ml extract load.

The concentrations of GITR were interpolated from the GITR standard curve and corrected for sample dilution. The mean GITR concentration was determined to be 37.69 ng/ml in HuT-102 cell extract. There was no detectable signal in Jurkat cell extract.

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

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