Mouse anti double-stranded RNA (J2, J5 and K1) Comparison Set

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)._x000D_ _x000D_ The J5 IgG2b antibody recognizes dsRNA with very similar affinity and specificity to our J2 antibody (see Schonborn et al., 1991), but has a different isotype – thus allowing more flexibility for the simultaneous detection of dsRNA with other markers, particularly in immunofluorescence microscopy, and has been used to detect replicative intermediates of the fish virus Infectious Pancreatic Necrosis Virus (IPNV) (Levican-Asenjo et al., 2019) or of ECMV in Vero cells._x000D_ The J5 antibody can detect all tested forms of dsRNA, including poly(A):poly(U), poly(I):poly(C) and dsRNA from viruses such as Dengue Virus, Encephalomyocarditis Virus, Vaccinia Virus, Reovirus or Cucumber Mosaic Virus. Similarly to our other antibodies dsRNA-binding of J5 is sequence-independent, as long as the length of the dsRNA exceeds 40nt. The antibody does not react with ssRNA, ssDNA or dsDNA. J5 has been tested successfully in nucleic acid ELISA, immunoblotting and immunofluorescence microscopy.

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.

Mouse monoclonal antibodies J2, J5 and K1 can be used for ELISA, dsRNA-immunoblotting, immunoaffinity chromatography and in certain systems also for immunohistochemistry (see references). The optimum working dilution of each antibody for any specific application should be established by titration._x000D_ _x000D_ Please note that nucleic acid separation prior to dsRNA-immunoblotting must be carried out by polyacrylamide gel electrophoresis, because the sensitivity of detection is considerably lower after blotting from agarose gels. _x000D_ _x000D_ Not for use for clinical purposes. For in vitro use only.

Mouse monoclonal antibody J2 recognises double-stranded RNA (dsRNA) provided that the length of the helix is greater than or equal to 40 bp. dsRNA-recognition is independent of the sequence and nucleotide composition of the antigen. All naturally occurring dsRNAs investigated up to; Now (40-50 species) as well as poly(I).poly(C) and poly(A).poly(U) have been recognised by J2, althoµgh in some assays its affinity to poly(I).poly(C) is about 10 times lower than that to other dsRNA antigens. Mouse monoclonal antibody J5 recognises double-stranded RNA (dsRNA) provided that the length of the helix is greater than or equal to 40 bp. dsRNA-recognition is independent of the sequence and nucleotide composition of the antigen. All naturally occurring dsRNAs investigated up to; Now (40-50 species) as well as poly(I).poly(C) and poly(A).poly(U) have been recognised by J5, althoµgh in some assays its affinity to poly(I).poly(C) is about 10 times lower than that to other dsRNA antigens. The mAb K1 recognises double-stranded RNA (dsRNA) provided that the length of the helix is greater than or equal to 40 bp. dsRNArecognition is independent of the sequence and nucleotide composition of the antigen. All naturally occurring dsRNAs investigated up to; Now (40-50 species) as well as poly(I).poly(C) and poly(A).poly(U) have been recognised by K1. As described by Schönborn et al. K1 shows higher affinity to poly(I).poly(C) than to the other dsRNA antigens, althoµgh the difference of apparent binding constants may vary under different experimental conditions. // The lyophilised samples should each be reconstituted with 100 µL sterile distilled water. The mAb will then be in PBS without any stabilisers or preservatives at a concentration of 1 mgr/mL. As a result of the lyophilisation procedure, the reconstituted antibody may contain small amounts of denatured protein in the form of aggregates that may interfere with some applications such as immunohistochemistry (e.g. by giving high backgrounds). We therefore highly recommend centrifuging (microcentrifuge) the reconstituted antibody before use and using the supernatant

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._x000D_ _x000D_ 9) Levicán-Asenjo J, Soto-Rifo R, Aguayo F, Gaggero A, Leon O. Salmon cells SHK-1 internalize infectious pancreatic necrosis virus by macropinocytosis. J Fish Dis. 2019 Jul;42(7):1035-1046. doi: 10.1111/jfd.13009._x000D_ _x000D_ _x000D_ Recent publication:_x000D_ Tirosh Shapira, I. Abrrey Monreal, Sébastien P Dion, Mason Jager, Antoine Désilets, Andrea D Olmstead, Thierry Vandal, David W Buchholz, Brian Imbiakha, Guang Gao, Aaleigha Chin, William D Rees, Theodore Steiner, Ivan Robert Nabi, Eric Marsault, Julie Sahler, Avery August, Gerlinde Van de Walle, Gary R Whittaker, Pierre-Luc Boudreault, Hector C Aguilar, Richard Leduc, François Jean. _x000D_ A novel highly potent inhibitor of TMPRSS2-like proteases blocks SARS-CoV-2 variants of concern and is broadly protective against infection and mortality in mice. bioRxiv 2021.05.03.442520; doi: https://doi.org/10.1101/2021.05.03.442520_x000D_ https://biorxiv.org/cgi/content/short/2021.05.03.442520v1

After reconstitution antibodies should be aliquoted and stored at -20 °C or -70°C; After adding 10 mM sodium azide undiluted antibody can also be stored at +4 °C for a short period of time; For long term storage the mAb should be kept frozen; Repeated freezing/thawing cycles should be avoided; When kept lyophilized the product will remain stable for 10 years at -20 °C or -70°C