Description
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.
HTRF assays offer many advantages over other technologies:
Homogeneous add-and-read format
No wash steps
Low background
Straightforward miniaturization from 96- or 384-well microplates to high density assay formats such as 384-well low volume and 1536-well plates
Stable signal, providing flexibility in time of readout or size of assays
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.
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.
