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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) [2] or by Toll-like Receptor 3 (TLR3) located to an endosomal-like compartment [3]. RIG-I and MDA5 belong to the family of RIG-I like receptor (RLR) together with the protein LGP2 whose function is still elusive [4, 5]. Upon binding to RNA, RIG-I and MDA5 bind a new mediator named Cardif, MAVS, IPS-1 or VISA [6, 7] that signals through transcription factors, such as IRF-3, IRF-7, and NF-κB leading to the activation of interferons (IFNs) and other inflammatory cytokine genes. RIG-I is activated by dsRNA and 5′-ppp RNA. RIG-I has been shown to trigger innate immune signaling pathways during infection by orthomyxoviruses, rhabdovirus, vesicular stomatitis virus, and paramyxoviruses; and possibly by the filovirus Ebola virus and by the Epstein-Barr virus [8]. Virus infection induces a rapid production of IFN-α/β that leads to expression of hundreds of interferon-stimulated genes (ISGs) whose products direct antiviral actions. RIG-I belongs to the ISG gene family and monitoring its levels using the RIG-I (human) Detection Set (IC) can be used as indicator of IFN production and virus infection.  RIG-I (human) Detection Set (IntraCellular)(APO-54N-038)Summary of features
Principle of the KitThis assay is a sandwich Enzyme Linked-Immuno-Sorbent Assay (ELISA) developed for the direct measurement of human RIG-I (hRIG-I) in cell extracts. A monoclonal antibody specific for hRIG-I (COAT) is coated onto the wells of the microtiter plate. Samples and standards of hRIG-I are pipetted into the wells for binding to the coated antibody. After extensive washing to remove unbound compounds, hRIG-I is recognized by the addition of a biotinylated polyclonal antibody specific for hRIG-I (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 hRIG-I in the samples.
Literature Overview:1. Takeuchi, O., and Akira, S. (2007). Recognition of viruses by innate immunity. Immun. Rev. 220, 214-224. 2. Takeuchi, O., and Akira, S. (2008). MDA5/RIG-I and virus recognition. Curr. Opin. Immunol. 20, 17-22. 3. Matsumoto, M., and Seya, T. (2008). TLR3: Interferon induction by double-stranded RNA including poly(I:C). Adv. Drug Deliv. Rev. 60, 805-812. 4. Yoneyama, M., Onomoto, K., and Fujita, T. (2007). Cytoplasmic recognition of RNA. Advanced drug delivery reviews 60, 841-846. 5. Venkataraman, T., Valdes, M., Elsby, R., Kakuta, S., Caceres, G., Saijo, S., Iwakura, Y., and Barber, G.N. (2007). Loss of DExD/H box RNA helicase LGP2 manifests disparate antiviral responses. J Immunol 178, 6444-6455. 6. Meylan, E., Curran, J., Hofmann, K., Moradpour, D., Binder, M., Bartenschlager, R., and Tschopp, J. (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., Takahashi, K., Sato, S., Coban, C., Kumar, H., Kato, H., Ishii, K.J., Takeuchi, O., and Akira, S. (2005). IPS-1, an adaptor triggering RIG-I- and Mda5-mediated type I interferon induction. Nat. Immunol. 6, 981-988. 8. Loo, Y.M., Fornek, J., Crochet, N., Bajwa, G., Perwitasari, O., Martinez-Sobrido, L., Akira, S., Gill, M.A., Garcia-Sastre, A., Katze, M.G., et al. (2008). Distinct RIG-I and MDA5 signaling by RNA viruses in innate immunity. J. Virol. 82, 335-345.
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