Human LAG3/MHC II biochemical binding assay
LAG3/MHC II biochemical binding assay
This immune checkpoint assay is designed to identify human CD47/SIRP alpha inhibitors.
Signal-regulatory protein alpha (SIRP-alpha), also known as CD172a, is a cell surface protein expressed mainly by myeloid cells, such as macrophages. Its receptor, CD47, is ubiquitously expressed on the surface of all cells and has been found to be overexpressed in some cancers. CD47 binding to SIRP-alpha delivers a "Do Not Eat Me" signal to macrophages. In the case of a tumor, this signaling aids in tumor evasion of the immune system. Inhibitors of this interaction hold great promise for cancer immunotherapy.
The CD47/SIRP binding assay includes human recombinant immune checkpoint partners tagged for detection (CD47 and SIRP alpha) and labelled anti-tag reagents for HTRF detection. Without an inhibitor, CD47 binds to SIRP alpha, 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 CD47/SIRP alpha interaction, the HTRF signal decreases.
This CD47/SIRP alpha 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 CD47 & SIRP alpha protein 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.
Performing an 8-point dose response curve using the blocking standard provided in the kit. The CD47-SIRP alpha interaction is inhibited, with an IC50 of 2.6 nM.
The inhibitory effects of three antibodies known to inhibit the CD47-SIRP alpha interaction were tested. The two anti-CD47 antibodies (B6H12 & B6H12.2) were shown to inhibit the interaction, with IC50s of 2.2 & 1.6 nM respectively. The anti-SIRP alpha antibody (SE5A5) disrupted the interaction, with an IC50 of 2.4 nM.
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