Description
IL6 is an inflammatory cytokine involved in various biological processes, including autoimmune diseases and cancers. It is produced by many cell lineages, including stromal cells, hematopoietic cells, epithelial cells, or muscle cells. IL6 binding to the membrane receptor IL6R induces receptor dimerization, and the activation of receptor-associated kinases (JAK1, JAK2, and Tyk2) which phosphorylate the receptor, leading to pathway stimulation. Anti-IL6 therapies have been developed for the treatment of various rheumatic diseases, as well as cytokine release syndrome.
HTRF assays offer many advantages over other technologies:
Homogeneous add-and-read format
No wash steps
Low background
Straightforward miniaturization from 96- or 384-well microplates to high density assay formats such as 384-well low volume and 1536-well plates
Stable signal, providing flexibility in time of readout or size of assays
Assay principle
The HTRF IL6/IL6 Binding kit is designed to measure the interaction between IL6 and IL6R proteins. Without treatment, IL6 binds to IL6R, and the binding of each detection antibody to its respective tagged protein generates an HTRF signal. In the presence of a compound or an anti-human blocking antibody, the IL6/IL6R interaction is disrupted and the HTRF signal decreases.
Assay protocol
The Human IL6/IL6R binding kit can be run in 96- or 384-well low volume white plates (20 µL final). As described here, samples or standards are dispensed directly into the assay plate. The Tag1-IL6 protein and Tag2-IL6R proteins are then added, followed by the dispensing of the HTRF reagents: the anti Tag1 antibody labeled with europium cryptate and the anti Tag2 antibody labeled with XL665. The reagents labeled 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.
Inhibitory effects of IL6/IL6R blockers
The inhibitory effects of unlabeled IL6, IL6R, Tocilizumab (anti IL6R), and Sarilumab (anti IL6) were tested. No inhibition was observed using an anti CD20 antibody (Rituxiamb), confirming the specificity of the kit for the study of IL6 / IL6R interactions.
