Double-stranded RNA (dsRNA) ELISA kit (K1 based)

Double-stranded RNA (dsRNA) ELISA kit (K1 based)

• Background: Based on the use of two double-stranded RNA (dsRNA)-specific monoclonal antibodies the dsRNA Detection Kit allows sensitive and selective detection of dsRNA molecules (larger than 30-40 bp), independent of their nucleotide composition and sequence. The detection is highly specific: dsRNA can be detected in nucleic acid extracts in the presence of 1.000-10.000-fold excess of other nucleic acids. This assay works on the sandwich-ELISA principle and uses the K1 (IgG2a) mouse monoclonal antibody to dsRNA as a catcher antibody. The monoclonal antibody K2 (IgM) is used as the detector antibody._x000D_ _x000D_ Over the past decade our double-stranded RNA (dsRNA)antibodies have been used extensively to detect and characterise plant and animal viruses with dsRNA genomes or intermediates. In addition, the anti-dsRNA antibodies can be used as a diagnostic tool to detect pathogens, including detection in paraffin-embedded fixed tissue samples (Richardson et al. 2010). _x000D_ The K1 monoclonal antibody recognises dsRNA with similar affinity to our widely used J2 antibody. It can be used for the histological and cytological detection of dsRNA in cells and tissues._x000D_ It has proven especially useful as an alternative to J2 to resolve cross-reactions and/or remove unwanted background, in those rare experimental setups where J2 did not provide satisfactory results._x000D_ K1 can be used to detect dsRNA intermediates of viruses as diverse as Hepatitis virus, Theiler’s murine encephalomyelitis virus or Japanese encephalitis virus. It has been for the detection of dsRNA in cultured cells and in fixed paraffin-embedded histological samples (see publications)._x000D_ If Poly I:C needs to be detected we highly using K1 rather than J2 because K1 has a much higher affinity for this synthetic polyribonucleotide (see Schönborn et al. 1991, Fig. 2)._x000D_ K1 has been used successfully in immunofluorescence microscopy, in flow cytometry (FACS) and in immunocapture methods (such as dot-blot and ELISA).
• Synonyms: dsRNA ELISA kit
• CAS Number: 7732-18-5
• Host: Mouse
• Isotype: IgG2a k, IgM k
• Clone: K1/K2
• Type: ELISA Kit
• Source: The two dsRNA antibodies used in this kit were produced as follows: Female DBA/2 mice were injected intraperitonially with a mixture of 50 ug L-dsRNA and 75 ug methylated bovine serum albumin, emulsified in complete Freund's adjuvant. After several boosts spleen cells were fused with Sp2/0-Agl4 myeloma cells to generate the hybridoma clones K1 and K2
• Applications: ELISA
• Field of Research: Infectious Agents and Disease, Microbiology
• Assay Principle: We recommend using the kit to detect viral dsRNAs or large natural or synthetic dsRNAs of non-viral origin in nucleic acid extracts, as well as to detect the presence of undesired dsRNA molecules in artificially synthesized (m)RNA preparations. Serial dilutions of the Poly(I:C) dsRNA standard (included in the kit) can be used as a positive control._x000D_ For the exact detection protocol we refer to the kit manual that can be downloaded from our website,_x000D_ _x000D_ Caution: The Poly(I:C) dsRNA positive control included in this sandwich ELISA kit is not intended to be used as a quantitative standard for other dsRNA preparations. The anti dsRNA antibodies K1 and/or K2 used in this kit may exhibit a different degree of reactivity with different dsRNAs obtained from synthetic or natural sources. It is therefore only intended to be used as a positive control to see if the ELISA has been executed correctly and that the test shows a linear relationship between the amount of dsRNA and the read out for the OD450.
• Form: Double-stranded RNA (dsRNA) ELISA kit containing the following reagents for 200 tests (2x96 wells):_x000D_ • 1 vial of coating antibody (store at -20 °C)_x000D_ • 1 vial of Poly(I:C) dsRNA as positive control (store at -20 °C)_x000D_ • 1 vial of dsRNA-specific detecting antibody (in RPMI + 5% FBS, store at +4 °C or, preferably, at -20 °C)_x000D_ • 1 vial of HRP-conjµgated F(ab’)2 Fragment of goat-anti mouse secondary antibody (store at +4 °C or at -20 °C)_x000D_ • 1 vial of TMB substrate solution (store at +4°C, keep in dark)
• References & Citations: 1) F. Weber, V. Wagner, S. B. Rasmussen, R. Hartmann, S. R. Paludan. Double-stranded RNA is produced by positive-strand RNA viruses and DNA viruses but not in detectable amounts by negative-strand RNA viruses. J Virol (2006), 80(10):5059-64. doi: 10.1128/JVI.80.10.5059-5064.2006._x000D_ _x000D_ 2) S. Welsch, S. Miller, I. Romero-Brey, A. Merz, C. K. E. Bleck, P. Walther, S. D. Fuller, C. Antony, J. Krijnse-Locker, R. Bartenschlager. Composition and Three-Dimensional Architecture of the Dengue Virus Replication and Assembly Sites. Cell Host , Microbe (2009) 5(4); 365-375. doi.org/10.1016/j.chom.2009.03.007. _x000D_ _x000D_ 3) K. Knoops , M. Bárcena, R. W. Limpens, A. J. Koster, A. M. Mommaas, E. J. Snijder. Ultrastructural characterization of arterivirus replication structures: reshaping the endoplasmic reticulum to accommodate viral RNA synthesis. J Virol. (2012) 86(5); 2474-2487. doi:10.1128/JVI.06677-11._x000D_ _x000D_ 4) S. J. Richardson, A. Willcox, D. A. Hilton, S. Tauriainen, H. Hyoty, A. J. Bone, A. K. Foulis, N. G. Morgan. Use of antisera directed against dsRNA to detect viral infections in formalin-fixed paraffin-embedded tissue. J Clin Virol. (2010) 49(3); 180-5. doi: 10.1016/j.jcv.2010.07.015._x000D_ _x000D_ 5) K. Karikó, H. Muramatsu, J. Ludwig, D. Weissman, Generating the optimal mRNA for therapy: HPLC purification eliminates immune activation and improves translation of nucleoside-modified, protein-encoding mRNA, Nucleic Acids Research (2011) 39(21); e142, https://doi.org/10.1093/nar/gkr695._x000D_ _x000D_ 6) Schönborn, J., Oberstrass, J., Breyel, E., Tittgen, J., Schumacher, J. and Lukacs, N. (1991) Monoclonal antibodies to double-stranded RNA as probes of RNA structure in crude nucleic acid extracts. Nucleic Acids Res.19, 2993-3000. _x000D_ _x000D_ 7) Lukacs, N. (1994) Detection of virus infection in plants and differentiation between coexisting viruses by monoclonal antibodies to double-stranded RNA. J. Virol. Methods 47, 255-272. _x000D_ _x000D_ 8) Lukacs, N. (1997) Detection of sense:antisense duplexes by structurespecific anti-RNA antibodies. In: Antisense Technology. A Practical Approach, C. Lichtenstein and W. Nellen (eds), pp. 281-295. IRL Press, Oxford.
• Shipping Conditions: Blue Ice
• Storage Conditions: Upon receipt, store entire kit at -20ºC; Once the kit is thawed, you may keep it at 4ºC for 5 days; For long-term storage, it is recommended to aliquot and freeze the antibody and dsRNA components at –20ºC