-->

Human PD1/PD-L1 biochemical binding assay HTRF®

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

See more
  • 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.

-
In stock

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

Best practices for pharmacological characterization of PPI inhibitors

Easy pharmacological characterization of PPI modulators. - Technical Notes

HTRF assays handle low- to high affinity protein-protein interactions

Deciphering low- and high affinity interactions - Application Notes

Nuclear receptor ligand identification with HTRF

Monitoring nuclear receptor binding with HTRF assays - Application Notes

HTRF addresses large protein-protein interaction complexes

Challenge large complexes with HTRF assays - Application Notes

HTRF PPI your dream assay served on a plate

Sandwiches aren't just for eating - Infographics

A brief history of Protein-Protein Interactions

How well do you know PPI? - Infographics

HTRF Product Catalog 2020 July update

All your HTRF assays in one document! - Catalog

A guide to Homogeneous Time Resolved Fluorescence

General principles of HTRF - Guides

How HTRF compares to Western Blot and ELISA

Get the brochure about technology comparison. - Brochures

Virology research solutions using HTRF Protein-Protein Interaction assays

See how peer researchers challenge the viral life cycle with PPI assays - Application Notes

Product Insert PD1/PD-L1 Kit2 / 64PD1PEG-64PD1PEH

64PD1PEG-64PD1PEH - Product Insert

autoimmune diseases therapeutic approaches

Complete autoimmunity review in a single White Paper - Guides

Autoimmune diseases and therapeutic strategies

Check out our new guide featuring valuable insights on autoimmunity - Guides

Plate Reader Requirement

Choosing the right microplate reader ensures you’ll get an optimal readout. Discover our high performance reader, or verify if your lab equipment is going to be compatible with this detection technology.

Let's find your reader