Human Osteopontin Recombinant Rabbit Monoclonal Antibody [PSH08-59] - BSA and Azide free (Detector)

Sandwich ELISA analysis of Human Osteopontin matched pair antibodies

Elisa assay was performed by coating wells of a 96-well plate with 100 µl per well of capture antibody (HA723004) diluted in carbonate/bicarbonate buffer, at a concentration of 5ug/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 Osteopontin protein (HA210993) starting from 5,000 pg/ml to 0 pg/ml and detect antibody (HA723005, 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 Osteopontin in human samples.

Interpolated concentration of native Osteopontin was measured in duplicate at different sample concentrations. The interpolated dilution factor corrected values were plotted (mean +/- SD, n=2). The mean Osteopontin concentration was determined to be 609 ng/mL in human urine, 27 ng/ml in U-87 MG cell culture supernatant. There was no detectable signal in Daudi cell supernatant.

Interpolated concentrations of spiked Osteopontin in cell culture media samples.

The concentrations of Osteopontin were measured in duplicates, interpolated from the Osteopontin 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=2).