This immune checkpoint assay is designed to identify human PD1/PD-L1 checkpoint inhibitors.

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  • All inclusive kit All inclusive kit
  • Scalable from 96 to 1536 format Scalable from 96 to 1536 format
  • Validated with reference Ab Validated with reference Ab

This immune checkpoint assay is designed to identify human PD1/PD-L1 checkpoint inhibitors.

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Overview

Programmed cell death protein 1 (PD1) is an immune checkpoint receptor that regulates T cell response. Its ligand, programmed death-ligand 1 (PD-L1), is commonly over-expressed on a tumor cell surface. When PD1 is bound to PD-L1, T cell response is suppressed, contributing to tumor immune resistance. Checkpoint inhibitors blocking PD1/PD-L1 complex formation are generating considerable interest in cancer immunotherapy.

Benefits

  • T CELL RESPONSE
  • IMMUNOTHERAPY TO FIGHT CANCER
  • DISCOVER IMMUNE CHECKPOINT BLOCKING DRUGS

Assay principle

The PD1/PD-L1 binding assay includes tagged human recombinant immune checkpoint partners  (PD1 and PD-L1) and labelled anti-tag reagents for HTRF detection. Without an inhibitor, PD1 binds to PD-L1, and the binding of each detection reagent to its tagged target generates an HTRF signal. In the presence of compound, standard, or an antibody blocking the PD1 /PD-L1 interaction, the HTRF signal decreases.

Principal of the HTRF human PD1/PD-L1 binding assay

Assay protocol

This PD1/PD-L1 binding assay can be run in a 96- or 384-well low volume white plate (20 µL final). As described here, samples or standards are dispensed directly into the assay plate, and the tagged PD1 & PD-L1 proteins are then added, followed by the dispensing of the HTRF reagents. The reagents labelled with HTRF fluorophores may be pre-mixed and added in a single dispensing step. No washing steps are needed. The protocol can be further miniaturized or upscaled by simply resizing each addition volume proportionally.

Protocol of the HTRF human PD1/PD-L1 binding assay

Inhibitory effect of blocking antibodies and small molecules

The inhibitory effects of three antibodies and two small molecules known to inhibit the PD1 / PD-L1 interaction were tested. The two anti-PD1 antibodies (pembrolizumab & nivolumab) were shown to inhibit the interaction with IC50 values of 2.0 & 2.4 nM respectively. The anti-PD-L1 antibody (atezolizumab) disrupted the interaction, with an IC50 of 3.9 nM. Two small molecule PD1 / PD-L1 inhibitors 1 & 3 show IC50s of 177 & 146 nM in these assay conditions.

Validation of the HTRF human PD1/PD-L1 binding kit Inhibitory effect of the blocking standard

Inhibitory effect of the PD1 / PD-L1 standard

The PD1 / PD-L1 standard stock solution provided in the kit was diluted to prepare the 8-point dose response curve using 5-fold serial dilutions. As described in the assay protocol, standards were dispensed into the assay plate, and the tagged PD1 & PD-L1 proteins were then added, followed by the dispensing of the HTRF reagents. The data presented in the graph show that the PD1 / PD-L1 interaction is inhibited, with an IC50 of 3.8 nM.




Validation of the HTRF human PD1/PD-L1 binding kit: Inhibitory effect of blocking antibodies and small molecules

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