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HTRF Human CTLA4/CD86 biochemical Binding Kit, 500 Assay Points
This immune checkpoint assay is designed to identify human CTLA4/CD86 checkpoint inhibitors.
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 & disposal requirements under European REACH regulations (EC 1907/2006).
Product Variants
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.
Specifications
| Assay Points |
500
|
|---|---|
| Assay Technology |
HTRF
|
| Brand |
HTRF
|
| Quantity |
1
|
| Therapeutic Area |
Oncology & Inflammation
|
| Unit Size |
500 Assay Points
|
Video gallery
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.
Resources
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