Description
Deregulation of the tumor necrosis factor (TNF) plays an important role in the
initiation and perpetuation of chronic inflammation and has been implicated in the development of various autoimmune diseases. TNFalpha inhibitors are successfully used for the treatment of several diseases, such as rheumatoid arthritis and psoriasis.
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 TNFα/TNFR1 Binding kit is designed to measure the interaction between TNFα and TNFR1 proteins. Without treatment, TNFa binds to TNFR1, 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 TNFa/TNFR1 interaction is disrupted and the HTRF signal decreases.
Assay protocol
The Human TNFalpha/TNFR1 binding kit 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. The Tag1-TNFalpha protein and Tag2-TNFR1 protein are then added, followed by the dispensing of the HTRF reagents: the Tag1 antibody labeled with europium cryptate and the Tag2 XL665 reagent. 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.
Inhibitory effects of TNFalpha/TNFR1 blockers
The inhibitory effects of small molecule unlabelled TNFR1 and TNFalpha, human anti-TNFalpha antibody (Adalimumab), and mouse anti-TNFR1 were tested. No inhibition was observed using an anti TNFR2 antibody, confirming the specificity of the kit for the study of TNFalpha/TNFR1 interaction.
