Description
Interleukin 17A, a pro-inflammatory cytokine, belongs to a family of six cytokines; IL-17A, IL-17B, IL-17C, IL-17D, IL-E, and IL-17F. IL-17A binds a heterodimeric cell surface receptor (IL-17RA/IL-17RC), triggering a signaling cascade that results in NF-κB activation which leads to the stimulation of the pro-inflammatory cytokines IL-6 and IL-8. Although this cytokine exerts a protective role against many pathogens, the overproduction of IL-17A is associated with autoimmune diseases such as psoriasis, rheumatid arthritis, and Crohn's Disease.
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 IL17A/IL17RA Binding kit is designed to measure the interaction between IL17A and IL17RA proteins. Without treatment, IL17A binds to IL17RA, 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 IL17A/IL17RA interaction is disrupted and the HTRF signal decreases.
Assay protocol
The Human IL17RA/IL17RA 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-IL17A protein and Tag2-IL17RA proteins are then added, followed by the dispensing of the HTRF reagents: the anti Tag1 reagent 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 IL17A/IL17RA blockers
The inhibitory effects of unlabeled IL17A and IL17R, and human anti-IL17A antibody, were tested. No inhibition was observed using an anti EGFR antibody (cetuximab), confirming the specificity of the kit for the study of IL17A/IL17RA interaction.
