Revvity, 64BDPIL12PEH, HTRF Human IL-23 / IL12R-β1 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 threatment 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 / IL12Rβ1 assay is highly specific for IL23 / IL12Rβ1, and hence is especially suitable for the discovery and qualification of anti p40 antibodies.
Assay principle The HTRF IL23/IL12Rβ1 Binding kit is designed to measure the interaction between IL23 and IL12Rβ1 proteins. Without treatment, IL23 binds to IL12Rβ1, 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/IL12Rβ1 interaction is disrupted and the HTRF signal decreases. Assay Protocol The Human IL23/IL2Rβ1 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-IL12Rβ1 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 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 IL23/IL12Rβ1 blockers The inhibitory effects of three antibodies against the p40 subunit of IL12/IL23 were tested, as well as the anti-IL6 & anti TNFa antibodies tocilizumab and adalimumab. Ustekinumab, MT3279H, and MT86/221 inhibit the interaction, with IC50's of 8.0, 1.5, and 4.6 nM respectively. As expected, Tocilizumab and adalimumab show no inhibition. Inhibitory effects of IL23/IL12Rβ1 standard A dose response curve for the standard using the conditions of the kit gives an IC50 of 1.4 ± 0.3 nM.