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HTRF Human CD47 / SIRP-α Binding Kit, 500 Assay Points

This immune checkpoint assay is designed to identify human CD47/SIRP alpha inhibitors.

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Feature	Specification
Application	Protein-Protein Interaction
Sample Volume	2 µL

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This immune checkpoint assay is designed to identify human CD47/SIRP alpha inhibitors.

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Product Variants

Part #:

64SIRPPEG

List Price

USD 861.02

Your online price:

Part #:

64SIRPPEH

List Price

USD 13,233.40

Your online price:

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, and disposal requirements under European REACH regulations (EC 1907/2006).

Product information

Overview

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.

Specifications

Other Specifications

Application	Protein-Protein Interaction
Brand	HTRF
Detection Modality	HTRF
Product Group	Kit
Sample Volume	2 µL
Shipping Conditions	Shipped in Dry Ice
Target Class	Binding Assay
Target Species	Human
Technology	TR-FRET
Therapeutic Area	Oncology & Inflammation
Unit Size	500 assay points

Video gallery

How it works

Assay principle

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.

Assay protocol

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.

Assay validation

Inhibitory effect of the blocking standard

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.

assay-validation-human-cd47-sirp-alpha-binding-kit-2

Inhibitory effect of blocking antibodies

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.