| Assay Method Information | |
| | Homogenouse Time-Resolved Fluorescence Assay |
| Description: | Homogeneous time-resolved fluorescence (HTRF) binding assay was used to determine the binding ability of the compound of the present disclosure to PD-1/PD-L1.The purchased kit (CisBio, #64CUS000C-1) contained reagents required for assays such as PD-1, PD-L1, anti-tag1-Eu, Anti-tag2-XL665, dilute buffer, and detection buffer.Experimental Procedure1. The compound was formulated to 10 concentrations with a 3-fold gradient with 100% DMSO.2. The DMSO solution of the compound was added to the dilute buffer, mixed thoroughly, then transferred to a 96-well plate.3. PD-L1 was diluted with dilute buffer, then added to the above 96-well plate.4. PD-1 was diluted with dilute buffer and added to the above 96-well plate, which was then incubated at room temperature for 30 minutes.5. A portion of anti-tag1-Eu and a portion of anti-tag2-XL665 were added to the detection buffer, mixed thoroughly and transferred to the above 96-well plate.6. The mixture in the above 96-well plate was incubated at room temperature for 1 to 24 hours.7. HTRF values were read with Envision. |
| Affinity data for this assay | |
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