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Human LAG3/MHCII biochemical binding assay HTRF®

This immune checkpoint assay is designed to identify human LAG3/MHC II 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 LAG3/MHC II checkpoint inhibitors.
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Overview

The lymphocyte activation gene-3 (LAG-3), selectively transcribed in human activated T and NK cells, encodes a ligand for major histocompatibility complex (MHC) class II molecules. When LAG3 is bound to MHC II, T cell response is suppressed. Checkpoint inhibitors blocking LAG3/MHC II complex formation are generating considerable interest in cancer immunotherapy.

Benefits

  • NO WASH BIOCHEMICAL FORMAT
  • EASY INHIBITOR CHARACTERIZATION
  • HTS COMPATIBLE

Assay principle

The LAG3/MHCII binding assay includes tagged human recombinant immune checkpoint partners  (LAG3 and MHCII) and labelled anti-tag reagents for HTRF detection. Without an inhibitor, LAG3 binds to MHCII, 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 LAG3/MHCII interaction, the HTRF signal decreases.

Principle of the HTRF Human LAG3/MHCII binding kit

Assay protocol

This LAG3/MHCII 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 LAG3 & MHCII 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.

Principle of the HTRF Human LAG3/MHCII binding kit

Inhibitory effect of blocking antibodies

The inhibitory effects of three antibodies against LAG3 and two non-specific antibdodies were tested. The two mAb anti-LAG3  were shown to inhibit the interaction with IC50 values of 1.1 & 2.2 nM respectively. The anti-LAG3 pAb inhibited the interaction with an IC50 of 27 nM whereas Nivolumab & Ipilimumab show no inhibition, as can be expected.

Compound screening on LAG3/MHCII binding kit

Inhibitory effect of the LAG3/MHCII standard

The LAG3/MHCII 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 LAG3 & MHCII proteins were then added, followed by the dispensing of the HTRF reagents. The data presented in the graph show that the LAG3 / MHCII interaction is inhibited, with an IC50 of 1.1 nM.

Dose response curve of the LAG3/MHCII standard

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Plate Reader Requirement

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