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METHOD VERIFICATIONPD-1 [BIOTINYLATED]:PD-L1 BINDING IN THE ABSENCE OF INHIBITORS:Immobilized human PD-L1 protein at 2 μg/mL (100 μL/well) can bind human PD-1-Biotin with a linear range of 0.01875-0.3 μg/mL when detected by Streptavidin-HRP. Background was subtracted from data points before curve fitting.INHIBITION OF PD-1 [BIOTINYLATED] : PD-L1 BINDING BY ANTI-PD-1 NEUTRALIZING ANTIBODYSerial dilutions of anti-PD-1 neutralizing antibody (1:2 serial dilutions, from 10 μg/mL to 0.078 μg/mL) was added into PD-L1 : PD-1-Biotin binding reactions. The assay was performed according to the above described protocol. Background was subtracted from data points prior to log transformation and curve fitting.Preparation of Standard Serial Dilutions1) Pipette 480 μL Dilution Buffer into tube Std-1 and 150 μL Dilution Buffer into the remaining tubes (tube Std.-2 to tube Std.-8).2) Add 20 μL anti-PD-1 neutralizing antibody stock solution (250 μg/mL) to tube Std.-1 to make the final concentration 10 μg/mL. Mix well.3) To make a 1:2 serial dilutions, pipette 150 μL solution to the next tube as illustrated in Fig.3.IMPORTANT: Mix thoroughly at each step during the dilution process.
Your experiment will include 4 simple steps: a) Coat the plate with human PD-L1, b) add your molecule of interest to the reactions, c) add biotinylated human PD-1 to bind with PD-L1, and d) add Streptavidin-HRP followed by TMB or other colorimetric HRP substrate to measure the binding activities. The ability of your compound to inhibit PD-1 : PD-L1 binding will be determined by comparing OD readings among different experimental groups.