Mouse PD-L1 Recombinant Rabbit Monoclonal Antibody [PSH04-83] - BSA and Azide free (Capture)

Sandwich ELISA analysis of Mouse PD-L1 matched pair antibodies

Elisa assay was performed by coating wells of a 96-well plate with 50 µl per well of capture antibody (HA722189) 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 Mouse PD-L1 protein (HA210689) starting from 5000 pg/ml to 0 pg/ml and detect antibody (HA722190, 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.

bEnd.3 cells were stimulated with 100 ng/ml mouse IFN-γ or vehicle control in 90%DMEM-H+10%FBS medium and incubated for 48 hours.The concentrations of PD-L1 were interpolated from the PD-L1 standard curves and corrected for sample dilution.Undiluted samples are mouse IFN-γ stimulated bEnd.3 cell extract 10% and unstimulated bEnd.3 cell extract 10%. The mean PD-L1 concentration was determined to be 26,524 pg/ml in mouse IFN-γ stimulated bEnd.3 cell extract and 1,337 pg/ml in the unstimulated bEnd.3 cell extract control.

Raw264.7 cells were stimulated with 100 ng/ml mouse IFN-γ or vehicle control in DMEM-H+10%FBS medium and incubated for 48 hours.The concentrations of PD-L1 were interpolated from the PD-L1 standard curves and corrected for sample dilution.Undiluted samples are mouse IFN-γ stimulated Raw264.7 cell extract 10% and unstimulated Raw264.7 cell extract 10%. The mean PD-L1 concentration was determined to be 11,723 pg/ml in mouse IFN-γ stimulated Raw264.7 cell extract and 471 pg/ml in the unstimulated Raw264.7 cell extract control.