Eurofins DiscoverX cytokine receptor assays are available as complete ready-to-use bioassay and eXpress kits and stable cell lines formats for receptor activation and dimerization assessment. Please contact custom assay development for additional assays. Learn more about the updated Cell Culture and Handling Procedure in this Technical Bulletin.
Assays for Cytokines Receptors
Eurofins DiscoverX cytokine receptors are based on the Enzyme Fragment Complementation (EFC) technology. These assays allow for the analysis of receptor activation and dimerization assessment through various assay modalities to ultimate allow for screening, potency and stability testing of therapeutic candidates. Select assay principles for receptor dimerization and IB protein degradation assays are shown below. For additional assay principles, please refer to receptor kinase, signaling pathway reporter, targeted protein degradation, and GPCR landing pages.
PathHunter® Tocilizumab Bioassay Assay Principle
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Measure IL-6 induced heterodimerization of IL-6 receptor (IL-6R) with the IL-6 Signal Transducer protein (IL-6ST; gp130) using the PathHunter Tocilizumab Bioassay (Cat. No.
93-1045B3-00110) qualified with the FDA approved therapeutic Actemra (tocilizumab, an anti-IL-6R antibody being evaluated as potential therapeutic for COVID-19). The assay principle involves the complementing of enzyme donor ProLink™ (PK) and enzyme acceptor (EA) EFC fragments tagged to IL-6ST and IL-6R, respectively. In presence of IL-6, the receptor units dimerize and there is an increase in signal detected upon substrate addition. However, in the presence of tocilizumab, the binding of IL-6 is inhibited and the signal decreases. Actemra® a registered trademark of Chugai Seiyaku Kabushiki Kaisha Corp., a member of the Roche Group.
PathHunter Adalimumab Bioassay Assay Principle
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Detect IkB protein degradation as a result of TNFα-mediated activation of the NF-kB signaling pathway using PathHunter Adalimumab Bioassay Kit (Cat. No.
93-0538B15-00132) qualified with the FDA approved therapeutic Humira (adalimumab), which is being evaluated as potential therapeutic for COVID-19. The assay principle involves the enzyme donor ProLabel® (PL) tagged to phosphorylated IkB, and the IκB levels can then be measured by the addition of EA, which forces complementation of the two EFC fragments. The resulting active enzyme hydrolyzes the substrate to generate the signal that is proportional to the degree of IκB stabilization. In presence of TNFα, which activates the NF-kB signaling pathway, proteasome-mediated IκB degradation occurs and there is a loss of signal. In the presence of both TNFα and adalimumab, which blocks the binding of TNFα, degradation of IκB is avoided, and the EFC complementation occurs leading to an increase in signal upon substrate addition. Humira® is a registered trademark AbbVie, Inc.
Analyze Multiple Interleukin Receptor Famillies with Highly Specific Assays
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Representative examples of assays for interleukin receptors from 6 different interleukin receptor families. Each plot shows a dose response for the relevant ligand(s) in a given assay from the indicated family. Data plotted are mean RLU and standard deviation from at least triplicate wells for each dose. These assays are characterized by robust assay windows and low CVs.
Qualify Therapeutics with MOA-Reflective, Receptor Dimerization Bioassays
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The PathHunter Anakinra Bioassay (Cat. No. 93-1032Y3-00106) was used to quantify an inhibitory response from Kineret (anakinra, an IL-1R antagonist being evaluated as potential therapeutic for COVID-19). [A.] In the presence of anakinra, there is a dose dependent inhibition (signal-to-background (S/B) 4.2; IC50 302 ng/mL) of IL-R1 and IL-1RAP dimerization. In comparison with agonist IL-1β, the receptors dimerize and there is an increase in signal detected (S/B 5.3; EC50 4.2 ng/mL). [B.] Dilutional linearity derived from the relative potency experiments performed over a range of 50-150%. Overall, the bioassay measures a robust response to both anakinra and IL-1β with a high accurate potency of over 99.4%. Kineret® is a registered trademark, licensed by Swedish Orphan Biovitrum AB and marketed by Sobi, Inc.
Accurately Measure Receptor Activation with Highly Reproducable Bioassays for QC Lot Release
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Tocilizumab Bioassay (Cat. No.
93-1045B3-00110) assessment of intra-day plate-to-plate and inter-day variability. Three experimental replicates of the assay were prepared as 11-point dose-response curves and run on the same plate with duplicate wells per dose on the same day by one operator. Results indicate 978, 960 and 997 ng/mL mean IC
50’s for plates 1-3, respectively, and an overall 9.7% intermediate precision IC
50. Similar experiments were repeated again to obtain data from three individual days (data not shown) for inter-day variability assessment with results showing an overall 1330 ng/mL mean IC
50 and 24.6% intermediate precision IC
50 for the 3 days. Results from both experiments for inter-plate and inter-day variability analysis show excellent uniformity demonstrating high reproducibility required for bioassay implementation in a QC environment for drug release.