Human CD47/SIRP alpha biochemical binding kit
CD47 / SIRP alpha biochemical binding assay
The lymphocyte activation gene-3 (LAG-3), selectively transcribed in human activated T and NK cells, encodes a ligand for major histocompatibility complex (MHC) class II molecules. When LAG3 is bound to MHC II, T cell response is suppressed. Checkpoint inhibitors blocking LAG3/MHC II complex formation are generating considerable interest in cancer immunotherapy.
The LAG3/MHCII binding assay includes tagged human recombinant immune checkpoint partners (LAG3 and MHCII) and labelled anti-tag reagents for HTRF detection. Without an inhibitor, LAG3 binds to MHCII, 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 LAG3/MHCII interaction, the HTRF signal decreases.
This LAG3/MHCII 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 LAG3 & MHCII 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 three antibodies against LAG3 and two non-specific antibdodies were tested. The two mAb anti-LAG3 were shown to inhibit the interaction with IC50 values of 1.1 & 2.2 nM respectively. The anti-LAG3 pAb inhibited the interaction with an IC50 of 27 nM whereas Nivolumab & Ipilimumab show no inhibition, as can be expected.
The LAG3/MHCII 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 LAG3 & MHCII proteins were then added, followed by the dispensing of the HTRF reagents. The data presented in the graph show that the LAG3 / MHCII interaction is inhibited, with an IC50 of 1.1 nM.
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