| Assay Method Information | |
| | Activity of Compounds to Inhibit PD-1/PD-L1 Interaction |
| Description: | Example 39: The experimental method and procedure were carried out in accordance with the kit instructions, which were briefly described as follows: The compounds were formulated into DMSO solutions of desired concentrations. Tagl-PD-L1, Tag2-PD-1 and compound solutions were diluted with diluent buffer to form working solutions, and anti-Tag1-Eu3+ and anti-Tag2-XL665 were diluted with detection buffer to form working solutions. White 384 shallow-well plates were used for the experiment, and 2 of compound working solution, 4 L of Tag1-PD-L1 working solution and 4 L of Tag2-PD-1 working solution were added in order to each well, and incubated at room temperature for 15 min; 10 L of a mixed solution formed by well mixing 5 L of anti-Tag1-Eu3+ working solution and 5 L of anti-Tag2-XL665 working solution was added to each well, and incubated for 2.5 h and detected; control groups were also set up in the experiment, wherein 2 L of diluent buffer was used to replace 2 L of compound working solution to form the Positive control, and 6 L of diluent buffer was used to replace 2 L of compound working solution and 4 L of Tag1-PD-L1 working solution to form the Negative control. Fluorescence intensities at emission wavelengths of 620 nm and 665 nm were detected under excitation light of 320 nm using Infinite F200 PRO from TECAN Company. HTRF value of each well=(fluorescence intensity at 665 nm/fluorescence intensity at 620 nm) 104, compound inhibition rate (%)=[1-(HTRF value of compound well HTRF value of Negative control well)/(HTRF value of Positive control well HTRF value of Negative control well)] 100%, and the inhibition rates of each compound at 8-10 concentrations were detected, and the IC50 was calculated using Prism software. |
| Affinity data for this assay | |
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