Human ICOSLG/B7-H2/CD275 Recombinant Rabbit Monoclonal Antibody [PSH11-33] - BSA and Azide free (Detector)

Sandwich ELISA analysis of human B7-H2 matched pair antibodies

Elisa assay was performed by coating wells of a 96-well plate with 50 µl per well of capture antibody (HA723317) diluted in carbonate/bicarbonate buffer, at a concentration of 1 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 ICOSLG/B7-H2/CD275 protein (HA210593) starting from 1,000 pg/ml to 0 pg/ml and detect antibody (HA723318, 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-H2 in JAR, U937 and Jurkat extract samples based on a 1,000 µg/ml extract load.

The concentrations of B7-H2 were measured in duplicates, interpolated from the B7-H2 standard curve and corrected for sample dilution. Undiluted samples are JAR extract 2.5%, U937 extract 100% and Jurkat extract 100%. The interpolated dilution factor corrected values are plotted (mean +/- SD, n=2). The mean B7-H2 concentration was determined to be 9,435 pg/ml in JAR extract, 1,040 pg/ml in U937 extract and undetectable in Jurkat extract.

Interpolated concentrations of spiked B7-H2 in human cell culture media samples.

The concentrations of B7-H2 were measured in duplicates, interpolated from the B7-H2 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).