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Human IL12/IL12Rβ1 binding kit HTRF®

The Human IL12/IL12Rβ1 binding kit is designed to identify human IL12/IL12Rβ1 inhibitors.

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  • No-wash No-wash
  • All inclusive kit All inclusive kit
  • Validated with reference Ab Validated with reference Ab

The Human IL12/IL12Rβ1 binding kit is designed to identify human IL12/IL12Rβ1 inhibitors.

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Overview

Also known as NKSF (natural killer cell stimulatory factor) and CLMF (cytotoxic lymphocyte maturation factor), IL12 is produced by macrophages and B cells. Along with IFN gamma, IL12 promotes the differentiation of naïve T cells into TH1 cells, which in turn release IFNg and IL2. Because of its effects on TH1 induction, IL12 is strongly linked to autoimmunity. IL12 is a heterodimer of p35 and p40 which binds the heterodimeric IL12Rβ1 , IL12Rβ2 complex.

The Human IL12/IL12Rβ1R assay is highly specific for IL12/IL12Rβ1 and hence is especially suitable for the discovery and characterization of anti p40 antibodies.

Benefits

  • AUTOIMMUNE DISEASE STUDIES
  • IL12/IL23 p40-BLOCKING DRUG DISCOVERY
  • KD DETERMINATION

Assay principle

The HTRF IL12/IL12Rβ1 Binding kit is designed to measure the interaction between IL12 and IL12Rβ1 proteins. Without treatment, IL12 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 IL12/IL12Rβ1 interaction is disrupted, and the HTRF signal decreases.

Principle of the HTRF Human IL12/IL12Rb1 binding kit

Assay Protocol

The Human IL12/IL12Rβ1 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-IL12 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.

Protocol of the HTRF IL12/IL12Rb1 assay

Inhibitory effects of IL12/IL12Rβ1 blockers

The inhibitory effects of three antibodies against the p40 subunit of IL12/IL23 were tested as well as two anti p19 (IL23) and the anti-IL6 & anti TNFa antibodies tocilizumab & adalimumab.  Ustekinumab, MT3279H & MT86/221 inhibit the interaction with IC50's of 1.6, 0.6 & 1.4 nM respectively. The two anti p19 (Guselkumab & MT155), tocilizumab & adalimumab show no inhibition.

Compound screening on IL12/IL12Rb1 assay

Inhibitory effects of IL12/IL12Rβ1 standard

A dose response curve for the standard using the conditions of the kit gives an IC50 of 0.8 ± 0.3 nM
Dose response  curve of the IL12-IL12Rb1 standard

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Plate Reader Requirement

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