Human CD73 Recombinant Rabbit Monoclonal Antibody [PSH08-16] - BSA and Azide free (Detector)

Sandwich ELISA analysis of human CD73 matched pair antibodies

Elisa assay was performed by coating wells of a 96-well plate with 50 µl per well of capture antibody (HA722957) 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 CD73 protein (HA210915) starting from 20,000 pg/ml to 0 pg/ml and detect antibody (HA722957, 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 CD73 in A431 extract samples based on a 1,000 µg/ml extract load.

The concentrations of CD73 were measured in duplicates, interpolated from the CD73 standard curve and corrected for sample dilution. Undiluted samples are A431 extract 25%. The interpolated dilution factor corrected values are plotted (mean +/- SD, n=2). The mean CD73 concentration was determined to be 6,201 pg/ml in A431 extract.

Interpolated concentrations of native CD73 in U-87 MG and Hela extract samples based on a 1,000 µg/ml extract load.

The concentrations of CD73 were measured in duplicates, interpolated from the CD73 standard curve and corrected for sample dilution. Undiluted samples are U-87 MG extract 13% and Hela extract 100%. The interpolated dilution factor corrected values are plotted (mean +/- SD, n=2). The mean CD73 concentration was determined to be 70,356 pg/ml in U-87 MG extract and undetectable in Hela extract.

Interpolated concentrations of spiked CD73 in human cell culture media samples.

The concentrations of CD73 were interpolated from the CD73 standard curves and corrected for sample dilution. Undiluted samples are as follows: cell culture media 1 50%, cell culture media 2 50%.