| Assay Method Information | |
| | Homogeneous Time-Resolved Fluorescence (HTRF) |
| Description: | PD1/PD-L1 binding assay kit (Cisbio, Cat #63ADK000CPDEC) was used, which includes two proteins, Tag 1-PD-L1 and Tag 2-PD-1, and two antibodies, Anti-Tag1-Eu3+ and Anti-Tag2-XL 665. Assay principle: Anti-tag1-Eu3+ is the HTRF donor, and Anti-Tag2-XL 665 is the HTRF acceptor. When Tag 1-PD-L1 and Tag 2-PD-1 interact, the added HTRF donor and the acceptor are close to each other. After the donor receives the excitation energy, it transfers part of the energy to the acceptor, thus producing 665 nm emission signal. When the compound is added to block the PD1/PD-L1 interaction, only 620 nm emission light was detected. The inhibitory effect of the compound was determined by analyzing the ratio of 665 nm/620 nm. Tag 1-PD-L1 was diluted with Diluent buffer (cat #62DLBDDF) to a working concentration of 10 nM, Tag 2-PD-1 was diluted with Diluent buffer to a working concentration of 500 nM, and Anti-Tag1-Eu3+ was diluted with detection buffer (cat #62DB1FDG) by 1:100, Anti-Tag2-XL 665 was diluted with detection buffer by 1:20, and the test compound was diluted with Diluent buffer to a final concentration of 2×. In a 384-well plate, 2 μL of compound was added to each well, and then 4 μL of Tag 1-PD-L1, 4 μL of Tag 2-PD-1 were added and incubated at room temperature for 15 minutes. |
| Affinity data for this assay | |
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