Double-stranded RNA (dsRNA) ELISA kit (K1 based)Catalog number: 10623002
|Isotype||IgG2a kappa/IgM kappa|
|Units||Reagents for 200 tests|
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. 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). 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. 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. 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). 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). 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
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
Double-stranded RNA (dsRNA) ELISA kit containing the following reagents for 200 tests (2x96 wells): •Reagent 01: 1 vial of coating antibody (store at -20 °C) • Reagent A: 1 vial of 142 bp dsRNA as positive contro(store at -20 °C) • Reagent B: 1 vial of dsRNA-specific detecting antibody (in RPMI + 5% FBS, store at +4 °C or, preferably, at -20 °C) • Reagent C: 1 vial of HRP-conjugated F(ab’)2 Fragment of goat-anti mouse secondary antibody (store at +4 °C or at -20 °C) • Reagent D: 1 vial of TMB substrate solution (store at +4°C, keep in dark)
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. By using serial dilutions of the 142 bp dsRNA standard (included in the kit) for calibration, quantitative estimates can also be made. Assay protocol: Materials and Equipment Required but Not Provided: • 2 ELISA plates (96 well) • Microtiter plate reader spectrophotometer with dual wavelength capability at 370 and 450 nm. • Single channel pipettes – 10 µl and 200 µl • Multichannel pipettes – 200 µl • Antigen (standard and sample) diluent (STE Buffer: 0.1 M NaCl, 1 mM EDTA, 50 mM Tris-HCl, pH 7.0) • Washing Buffer (PBS + 0.5% Tween 20; PBS: 10 mM Pi-buffer, pH 7.2, 0.15 M NaCl) • Secondary antibody dilution buffer (PBS+1% BSA) • Incubator allowing incubation at 37 °C. • 2 M H2SO4 Preparation of Reagents and ELISA Plates • Reconstitute Reagent A by adding 20 µl RNase-free MilliQ water. The concentration will be 1 µg/µl dsRNA. (Store it at -20 °C or -80 °C.) • Use DEPC treated MilliQ water to prepare STE (when applicable for your own sample preparation) • Sterilize PBS and STE by autoclaving • Prepare PBS + 1% BSA and ELISA washing buffer Coating of ELISA plates • Transfer the total content of the Reagent 02 tube into 21 ml PBS, mix well and immediately distribute 100 l/well in 2 ELISA plates. • Cover the plates and incubate them overnight at 4 C. • Discard contents of wells into waste. Add 100 l/well 1% BSA in PBS + 0.2% NaN3 to each well and incubate at 37 C for 2 h to saturate any remaining free binding sites on the plate. • Discard the solution and wash plates 3 times with PBS + 0.5% Tween 20. • The plates can then be used directly or stored. For storage fill the wells with 200 l/well PBS containing 0.2% sodium azide. Wrap plates in plastic foil and store them refrigerated at 4 C. They can be stored without any loss of activity for one month. When stored plates are used, they must be thoroughly washed with PBS to remove all traces of NaN3. Assay Protocol for 1 plate • All standards and samples should be assayed at least in duplicate. • Use clean, RNase-free micro-centrifuge tubes with cap. • Do not use buffers which contain NaN3 as it will interfere with the final detection step. 1. Prepare 1:3 serial dilutions from Reagent A. The dilution series of the dsRNA standard should be in the range of expected dsRNA concentration of your sample. We propose starting with 10 ng dsRNA/well as the highest concentration and diluting down to below 0.01 ng dsRNA/well. Dilutions should be freshly made for each assay. 2. Prepare dilutions of your sample in STE (when necessary). 3. Cap and vortex all diluted standards and samples. 4. Remove the plastic foil from the ELISA plate, discard the liquid and wash twice with PBS + 0.5 % Tween 20. 5. Discard the solution and transfer 100-100 µl antigen to duplicated wells in the plate. 6. Cover and incubate for 60 min at 37 °C. 7. Discard contents of wells into waste. Wash plate 4 times with PBS + 0.5 % Tween 20 adding 250 µl washing solution/well. Do not allow wells to dry before adding the next solution. 8. Pipette 100 µl undiluted Reagent B into all wells. 9. Cover and incubate for 60 minutes at 37 °C. 10. During the incubation (step 9) dilute Reagent C by pipetting 1.6 µl Reagent C into 10 ml PBS + 1% BSA (no azide!!!). 11. Discard content of ELISA plate into waste and wash as in Step 7. 12. Add 100 µl diluted Reagent C to each well. 13. Cover and incubate for 60 minutes at 37 °C. 14. Wash as in Step 7. Take care to remove all washing fluid after the last wash. 15. Pipette 100 µl undiluted Reagent D into each well. 16. Cover and incubate in the dark at room temperature (20±5 °C). 17. Read absorbance at adequate intervals at 370 nm blanking on the Zero standard (no dsRNA antigen). (We usually read between 5 - 60 min.) 18. When the absorbance has reached the optimum level stop reaction by adding 100 µl of 2M H2SO4 to all wells. Read absorbance at 450 nm blanking on the Zero Standard.
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.
Shipping Conditions: The Double-stranded RNA (dsRNA) ELISA kit components are shipped on blue ice.
This product is intended FOR RESEARCH USE ONLY, and FOR TESTS IN VITRO, not for use in diagnostic or therapeutic procedures involving humans or animals. It may contain hazardous ingredients. Please refer to the Safety Data Sheets (SDS) for additional information and proper handling procedures. Dispose product remainders according to local regulations.This datasheet is as accurate as reasonably achievable, but Exalpha Biologicals accepts no liability for any inaccuracies or omissions in this information.
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. 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. 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. 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. 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. 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. 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. 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.
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