Human CD276 Recombinant Rabbit Monoclonal Antibody [PSH05-92] - BSA and Azide free (Capture)

Sandwich ELISA analysis of Human CD276 matched pair antibodies

Elisa assay was performed by coating wells of a 96-well plate with 50 µl per well of capture antibody (HA722491) 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 CD276 protein (HA210964) starting from 2,000 pg/ml to 0 pg/ml and detect antibody (HA722491, Biotin, 0.1 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 B7-H3 in human serum and human urine samples.

The concentrations of B7-H3 were measured in triplicates, interpolated from the B7-H3 standard curve and corrected for sample dilution. Undiluted samples are human serum 5% and human urine 10%. The interpolated dilution factor corrected values are plotted (mean +/- SD, n=3). The mean B7-H3 concentration was determined to be 19.1ng/ml in human serum and 4.9 ng/ml in human urine.

Interpolated concentrations of native B7-H3 in A431 and NIH:OVCAR-3 cell culture supernatant.

The concentrations of B7-H3 were measured in triplicates, interpolated from the B7-H3 standard curve and corrected for sample dilution. Undiluted samples are A431 cell culture supernatant 100% and NIH:OVCAR-3 cell culture supernatant 100%. The interpolated dilution factor corrected values are plotted (mean +/- SD, n=3). The mean B7-H3 concentration was determined to be 199.5 pg/ml in A431 cell culture supernatant and 476.8 pg/ml in NIH:OVCAR-3 cell culture supernatant.

Interpolated concentrations of spiked B7-H3 in human cell culture media samples.

The concentrations of B7-H3 were measured in triplicates, interpolated from the B7-H3 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).