| Assay Method Information | |
| | HTRF Assay |
| Description: | As further confirmation of the results of the NMR assay and to classify the compounds of the invention based on their in vitro ability to inhibit PD-1/PD-L1 interaction, a homogeneous fluorescence binding assay resolved over time (HTRF) was used. This assay allows a simple and rapid characterisation of the inhibitors in a high-throughput format. Basically, it uses labelled human recombinant immune checkpoint partners (hPD1 and hPD-L1) and labelled anti-tag reagents for HTRF detection. In more detail, the interaction between hPD-L1 (Tag 1) and hPD1 (Tag2) is detected using Europium-labelled anti-Tag1 (HTRF donor) and XL665-labelled anti-Tag2 (HTRF acceptor). After hPD-L1 binds hPD1, the donor and acceptor antibodies are in close proximity, so excitation of the donor antibody triggers fluorescence resonance energy transfer (FRET) towards the acceptor antibody, which in turn specifically emits at 665 nm. This signal is directly proportional to the extent of the hPD1/hPD-L1 interaction. Thus, the compounds capable of inhibiting the PD1/PD-L1 interaction induce a reduction in HTRF signal. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |