Human CTLA4/CD80 biochemical binding assay HTRF®

This immune checkpoint assay is designed to identify human CTLA4/CD80 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 CTLA4/CD80 checkpoint inhibitors.

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Overview

CTLA4 (cytotoxic T-lymphocyte-associated protein 4), also known as CD152, is an inhibitor of the immune response expressed by T cells. CTLA4 and CD28 (stimulatory checkpoints) share the same ligands, namely CD80 (B7.1) and CD86 (B7.2), with CTLA4 displaying a greater affinity than CD28 for both, thus creating effective ligand binding competition. Although CTLA4 can be found on all T cells, it is mainly expressed on Treg (regulatory T cells), where it participates in their inhibition of the immune response.

Benefits

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

Assay principle

The CTLA4/CD80 binding assay includes tagged human recombinant immune checkpoint partners  (CTLA4 and CD80 (or B7.1)) and labelled anti-tag reagents for HTRF detection. Without an inhibitor, CTLA4 binds to CD80, 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 CTLA4 /CD80 interaction, the HTRF signal decreases.

Assay principle of the HTRF CTLA4/CD80 binding kit

Assay protocol

This CTLA4/CD80 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 CTLA4 & CD80 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 protocol of the HTRF CTLA4/CD80 binding kit

Inhibitory effect of blocking antibodies

The inhibitory effects of seven antibodies against CTLA4, CD80 , CD86, and PD-1 were tested. The two anti-CTLA4 antibodies (Ipilimumab & L3D10) were shown to inhibit the interaction, with IC50 values of 1.1 & 2.7 nM respectively. The anti-CD80 antibodies (MAB140 & 2D10) disrupted the interaction, with IC50s of 0.30 & 11.5 nM respectively. Anti-CD86 antibodies & nivolumab (anti-PD1) show no inhibition, as could be expected.

Assessment of 7 antibodies using the HTRF CTLA4/CD80 binding kit

Inhibitory effect of the CTLA4/CD80 standard

The CTLA4 / CD80 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 CTLA4 & CD80 proteins were then added, followed by the dispensing of the HTRF reagents. The data presented in the graph show that the CTLA4 / CD80 interaction is inhibited, with an IC50 of 1.0 nM.

Dose response curve of the CTLA4/CD80 standard using the HTRF CTLA4/CD80 binding kit

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