Revvity, 64BDPIL23PEH, HTRF Human IL-23 / IL23R Binding Kit, 10,000 Assay Points

IL23 is part of the response against infection, as it upregulates the expression of MMP9 (matrix metalloprotease 9), increases angiogenesis, and reduces CD8 T cell infiltration into tumors. In oncology and immuno-oncology, IL23 has been studied for its effects on T cell infiltration and its promotion on TH17 cell development. Anti IL23 antibodies have also been developed for the treatment of Crohn's Disease and psoriasis. IL23 is a heterodimeric cytokine composed of p19 and p40 subunits, and which binds the heterodimeric IL23R, IL12Rβ1 complex. The Human IL23-IL23R assay is highly specific for IL23/IL23R, and hence is especially suitable for the discovery and characterization of anti p19 antibodies.
Assay principle The HTRF IL23/IL23R Binding kit is designed to measure the interaction between IL23 and IL23R proteins. Without treatment, IL23 binds to IL23R, 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 IL23/IL23R interaction is disrupted, and the HTRF signal decreases. Assay Protocol The Human IL23/IL23R 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-IL23 and Tag2-IL23R proteins are then added, followed by the dispensing of the HTRF reagents: the anti Tag1 antibody labeled with europium cryptate, and the anti Tag2 reagent labeled with d2. 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 IL23/IL23R blockers The inhibitory effects of two antibodies against the p19 subunit of IL23 were tested. Guselkumab & MT155 were shown to inhibit the interaction, with both having IC50 of 2.5 nM. The anti-IL6 and anti TNFa antibodies (tocilizumab & adalimumab respectively) show no inhibition, as could be expected. Inhibitory effect of the IL23/IL23R standard A dose response curve for the standard using the conditions of the kit gives an IC50 of 1.6 ± 0.1 nM.