Human CD47/SIRP alpha biochemical binding kit
CD47 / SIRP alpha biochemical binding assay
-->
This immune checkpoint assay is designed to identify human CTLA4/CD80 checkpoint inhibitors.
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/CD80 binding assay includes tagged human recombinant immune checkpoint partners (CTLA4 and CD80 (or B7.1)) and labelled anti-tag reagents for HTRF detection. Without an inhibitor, CTLA4 binds to CD80, 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 /CD80 interaction, the HTRF signal decreases.
This CTLA4/CD80 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 & CD80 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, CD80 , CD86, and PD-1 were tested. The two anti-CTLA4 antibodies (Ipilimumab & L3D10) were shown to inhibit the interaction, with IC50 values of 1.1 & 2.7 nM respectively. The anti-CD80 antibodies (MAB140 & 2D10) disrupted the interaction, with IC50s of 0.30 & 11.5 nM respectively. Anti-CD86 antibodies & nivolumab (anti-PD1) show no inhibition, as could be expected.
The CTLA4 / CD80 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 & CD80 proteins were then added, followed by the dispensing of the HTRF reagents. The data presented in the graph show that the CTLA4 / CD80 interaction is inhibited, with an IC50 of 1.0 nM.
Best practices for pharmacological characterization of PPI inhibitors
Easy pharmacological characterization of PPI modulators. - Technical Notes
HTRF assays handle low- to high affinity protein-protein interactions
Deciphering low- and high affinity interactions - Application Notes
Nuclear receptor ligand identification with HTRF
Monitoring nuclear receptor binding with HTRF assays - Application Notes
HTRF addresses large protein-protein interaction complexes
Challenge large complexes with HTRF assays - Application Notes
HTRF PPI your dream assay served on a plate
Sandwiches aren't just for eating - Infographics
A brief history of Protein-Protein Interactions
How well do you know PPI? - Infographics
All your HTRF assays in one document! - Catalog
A guide to Homogeneous Time Resolved Fluorescence
General principles of HTRF - Guides
How HTRF compares to Western Blot and ELISA
Get the brochure about technology comparison. - Brochures
Virology research solutions using HTRF Protein-Protein Interaction assays
See how peer researchers challenge the viral life cycle with PPI assays - Application Notes
Autoimmune diseases and therapeutic strategies
Check out our new guide featuring valuable insights on autoimmunity - Guides
Assays for autoimmunity research
Advance your research on autoimmune diseases - Flyers
Manual CtlA4/CD80 Kit / 64CTLA80PEG-64CTLA80PEH
64CTLA80PEG-64CTLA80PEH - Product Insert
Certificate of Analysis CtlA4/CD80 Kit / 64CTLA80PEG
64CTLA80PEG Batch 02C - Quality Control Report
Safety Data Sheet (DEU) CtlA4/CD80 Kit / 64CTLA80PEG
64CTLA80PEG - Safety Data Sheet
Safety Data Sheet (ELL) CtlA4/CD80 Kit / 64CTLA80PEG
64CTLA80PEG - Safety Data Sheet
Safety Data Sheet (FRA-FR) CtlA4/CD80 Kit / 64CTLA80PEG
64CTLA80PEG - Safety Data Sheet
Safety Data Sheet (ITA) CtlA4/CD80 Kit / 64CTLA80PEG
64CTLA80PEG - Safety Data Sheet
Safety Data Sheet (SPA) CtlA4/CD80 Kit / 64CTLA80PEG
64CTLA80PEG - Safety Data Sheet
Safety Data Sheet (ENG-GB) CtlA4/CD80 Kit / 64CTLA80PEG
64CTLA80PEG - Safety Data Sheet
Safety Data Sheet (ENG-US) CtlA4/CD80 Kit / 64CTLA80PEG
64CTLA80PEG - Safety Data Sheet
Safety Data Sheet (DEU) CtlA4/CD80 Kit / 64CTLA80PEH
64CTLA80PEH - Safety Data Sheet
Safety Data Sheet (ELL) CtlA4/CD80 Kit / 64CTLA80PEH
64CTLA80PEH - Safety Data Sheet
Safety Data Sheet (FRA-FR) CtlA4/CD80 Kit / 64CTLA80PEH
64CTLA80PEH - Safety Data Sheet
Safety Data Sheet (ITA) CtlA4/CD80 Kit / 64CTLA80PEH
64CTLA80PEH - Safety Data Sheet
Safety Data Sheet (SPA) CtlA4/CD80 Kit / 64CTLA80PEH
64CTLA80PEH - Safety Data Sheet
Safety Data Sheet (ENG-GB) CtlA4/CD80 Kit / 64CTLA80PEH
64CTLA80PEH - Safety Data Sheet
Safety Data Sheet (ENG-US) CtlA4/CD80 Kit / 64CTLA80PEH
64CTLA80PEH - Safety Data Sheet
Choosing the right microplate reader ensures you’ll get an optimal readout. Discover our high performance reader, or verify if your lab equipment is going to be compatible with this detection technology.
Let's find your readerYou might be interested in