Human IL-2 Receptor alpha Recombinant Rabbit Monoclonal Antibody [PSH05-66] - BSA and Azide free (Detector)

Sandwich ELISA analysis of human IL2RA matched pair antibodies

Elisa assay was performed by coating wells of a 96-well plate with 100 µl per well of capture antibody (HA722385) 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 IL2RA protein (HA210771) starting from 2,000 pg/ml to 0 pg/ml and detect antibody (HA722386, 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 IL2RA in human PBMC cell culture supernatant.

PBMC cells were stimulated with 10 µg/ml PHA-M or vehicle control and incubated for 2 days. The concentrations of IL2RA measured in duplicate and interpolated from the IL2RA standard curve and corrected for sample dilution. Undiluted samples are as follows: unstimulated 100% and stimulated 50%. The interpolated dilution factor corrected values are plotted (mean +/- SD, n=2). The mean IL2RA concentration was determined to be 1,306.5 pg/ml in PHA-M stimulated PBMC cell culture supernatant and 101.3 pg/ml in the unstimulated PBMC control.

Interpolated concentrations of native IL2RA in human serum samples.

The concentrations of IL2RA were measured in triplicates, interpolated from the IL2RA standard curve and corrected for sample dilution. Undiluted samples are human serum 2.5%. The interpolated dilution factor corrected values are plotted (mean +/- SD, n=3). The mean IL2RA concentration was determined to be 3,193 pg/mL in human serum.

Interpolated concentrations of spiked IL2RA in human cell culture media samples.

The concentrations of IL2RA were measured in triplicates, interpolated from the IL2RA 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=3).