| Assay Method Information | |
| | In Vitro Inhibitory Activity Against PD-1/PD-L1 Binding |
| Description: | The experiment process was carried out according to the flow required in the operating manual of the test reagent.The process was as follows:(1) Experiment preparation: A test compound was diluted to different concentration gradients with the dilution buffer (the highest final concentration in a 20 μL final reaction system was 10 μM). The His-PD-1 protein was diluted to 800 nM (the final concentration in the 20 final reaction system was 100 nM). The PD-L1-Fc fusion protein was diluted to 16 nM (the final concentration was 2 nM). The anti-His-XL665 antibody and the anti-hFc-Eu3+ antibody were diluted 20 times and 100 times respectively with the detection buffer according to reagent requirements.(2) First, 5 μL of the test compound, 2.5 μL of the PD-L1-Fc fusion protein and 2.5 μL of the His-PD-1 protein solution were well mixed and incubated at room temperature for 15 min; then, 5 μL of the anti-His-XL665 antibody and 5 μL of the anti-hFc-Eu3+ antibody were added to the system and further incubated for 3 h before test.(3) During the test reaction, control groups were set, including a 0% inhibition positive control without adding the test compound and a 100% inhibition negative control without adding the PD-1 protein. All tests were conducted by use of multiple holes.(4) The fluorescence detector Tecan (Spark 10M) was used to detect the fluorescence signal of each hole. The excitation wavelength was 320 nm, and the emission wavelengths for detection were 620 nm and 665 nm, respectively. The strength of PD-1/PD-L1 binding refers to a fluorescence signal ratio Em665/Em620.(5) Calculation formula of binding inhibition rate of test compound: Inhibition rate (%)=[1−(fluorescence signal ratio of detected hole−fluorescence signal ratio of 100% inhibition negative control)]/(fluorescence signal ratio of 0% inhibition positive control−fluorescence signal ratio of 100% inhibition negative control)×100%. The 50% inhibition concentration (IC50) was calculated after the PD-1/PD-L1 binding inhibition rates of the test compound with different concentration gradients were calculated respectively. |
| Affinity data for this assay | |
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