Human LAG3/MHC II biochemical binding assay
LAG3/MHC II biochemical binding assay
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.
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.
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.
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.
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.
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