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RIG-I-like receptors (RLRs)The innate immune response to viruses is initiated upon detection of viral RNA generated during the life cycle of most viruses [1]. Recognition of the viral RNA is mediated by either the two cytoplasmic RNA helicases Retinoic acid Inducible Gene-1 (RIG-I ; Ddx58) and Melanoma Differentiation Associated gene 5 (MDA5 / Helicard; Ifih1) [2] or by Toll-like Receptor 3 (TLR3) located to an endosomal-like compartment [3]. RIG-I and MDA5 belongs to the family of RIG-I-like receptors (RLRs) together with the protein LGP2 whose function is still elusive [4, 5]. Upon binding to RNA, RIG-I or MDA5 binds a new mediator named Cardif, MAVS (mitochondrial antiviral signalling), IPS-1 (interferon-beta; promoter stimulator 1) or VISA (virus-induced signalling adapter) [6, 7]. Cardif/ MAVS/ IPS-1/ VISA then interacts with several TRAFs molecules, leading to the activation of regulatory kinases, such as TBK1, IKKepsilon; , and the IKK complex. These kinases activate respective transcription factors, IRF-3, IRF-7, and NF-kB that participate in the activation of IFN and other inflammatory cytokine genes. RIG-I is activated by dsRNA and 5'-ppp RNA while only poly I:C acts as a chemical agonist for MDA5. The physiological agonist of MDA5 has still to be discovered. Mda-5 is an early response gene inducible by IFN and tumor necrosis factor-alpha, responding predominantly to IFN-beta [8].  MDA5 (human) Detection Set (IntraCellular)(APO-54N-035)Summary of features
Principle of the KitThis assay is a sandwich Enzyme Linked-Immuno-Sorbent Assay (ELISA) developed for the direct measurement of human MDA5 (hMDA5) in cell extracts. A polyclonal antibody specific for hMDA5 (COAT) is coated onto the wells of the microtiter plate. Samples and standards of hMDA5 are pipetted into the wells for binding to the coated antibody. After extensive washing to remove unbound compounds, hMDA5 is recognized by the addition of a biotinylated monoclonal antibody specific for hMDA5 (DET). After removal of excess biotinylated antibody, streptavidine-peroxidase is added. Following a final washing, peroxidase activity is quantified using the substrate 3,3’,5,5’-tetramethylbenzidine (TMB). The intensity of the color reaction is measured at 450 nm after acidification and is directly proportional to the concentration of hMDA5 in the samples.
Literature Overview:1. Takeuchi, O., and Akira, S. (2007). Recognition of viruses by innate immunity. Immunol. Rev. 220, 214-224. 2. Takeuchi, O., and Akira, S. (2008). MDA5/RIG-I and virus recognition. Curr. Opinion in Immunol. 20, 17-22. 3. Matsumoto, M., and Seya, T. (2008). TLR3: Interferon induction by double-stranded RNA including poly(I:C). Advanced drug delivery reviews. [Epub ahead of print]. 4. Yoneyama, M., Onomoto, K., and Fujita, T. (2007). Cytoplasmic recognition of RNA. Advanced drug delivery reviews. [Epub ahead of print]. 5. Venkataraman, T.,et al. (2007). Loss of DExD/H box RNA helicase LGP2 manifests disparate antiviral responses. J Immunol .178, 6444-6455. 6. Meylan, E., et al. (2005). Cardif is an adaptor protein in the RIG-I antiviral pathway and is targeted by hepatitis C virus. Nature 437, 1167-1172. 7. Kawai, T., et al. (2005). IPS-1, an adaptor triggering RIG-I- and Mda5-mediated type I interferon induction. Nat. Immunol.. 6, 981-988. 8. Kang, D.C., Gopalkrishnan, R.V., Wu, Q., Jankowsky, E., Pyle, A.M., and Fisher, P.B. (2002). mda-5: An interferon-inducible putative RNA helicase with double-stranded RNA-dependent ATPase activity and melanoma growth-suppressive properties. PNAS. 99, 637-642.
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