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HTRF Human CTLA4 / CD86 Binding Kit, 500 Assay Points

Generic HTRF biochemical binding assay XL665 image

This immune checkpoint assay is designed to identify human CTLA4/CD86 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 CTLA4/CD86 checkpoint inhibitors.

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Product variants

Unit Size: 500 assay points

Part #:

64CTLA86PEG

Unit Size: 10,000 assay points

Part #:

64CTLA86PEH

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

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.

How it works

Assay principle

The CTLA4/CD86 binding assay includes tagged human recombinant immune checkpoint partners (CTLA4 and CD86 (or B7.2)) and labelled anti-tag reagents for HTRF detection. Without an inhibitor, CTLA4 binds to CD86, 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 /CD86 interaction, the HTRF signal decreases.

Assay protocol

This CTLA4/CD86 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 & CD86 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

The inhibitory effects of seven antibodies against CTLA4, CD86 , CD80, and PD-1 were tested. The two anti-CTLA4 antibodies (Ipilimumab & L3D10) were shown to inhibit the interaction, with IC50 values of 1.1 & 6.9 nM respectively. The anti-CD86 antibodies (MAB141 & biolegend 305402) disrupted the interaction, with IC50s of 0.92 & 0.68 nM respectively. Anti-CD80 antibodies & nivolumab (anti-PD1) show no inhibition, as could be expected.

Inhibitory effect of the CTLA4/CD86 standard

The CTLA4 / CD86 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 & CD86 proteins were then added, followed by the dispensing of the HTRF reagents. The data presented in the graph show that the CTLA4 / CD86 interaction is inhibited, with an IC50 of 1.0 nM.

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