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HTRF Human PD1 / PDL1 Binding Kit, 500 Assay Points

Generic HTRF sadwich assay Tb reagent XL665 Ab image

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

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Feature	Specification
Application	Protein-Protein Interaction
Sample Volume	2 µL

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

View product information

Product variants

Unit Size: 500 assay points

Part #:

64PD1PEG

Unit Size: 10,000 assay points

Part #:

64PD1PEH

For research use only. Not for use in diagnostic procedures. All products to be used in accordance with applicable laws and regulations including without limitation, consumption, and disposal requirements under European REACH regulations (EC 1907/2006).

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Product information

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.

How it works

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.

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.

Assay validation

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.

assay-validation-human-pd1-pdl1-binding-kit-1

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.

assay-validation-human-pd1-pdl1-binding-kit-2

Specifications

Other specifications

Application	Protein-Protein Interaction
Brand	HTRF
Detection Modality	HTRF
Product Group	Kit
Sample Volume	2 µL
Shipping Conditions	Shipped in Dry Ice
Target Class	Binding Assay
Target Species	Human
Technology	TR-FRET
Therapeutic Area	Oncology & Inflammation
Unit Size	500 assay points