KRIBIOLISA™ Double-Stranded RNA (dsRNA) ELISA (J2 based)

Introduction:

The J2 anti-dsRNA IgG2a monoclonal antibody (Schönborn et al. 1991) has become the gold standard in dsRNA detection. It was used initially for the study of plant viruses, but since the seminal paper of Weber et al.In 2006, where J2 was used to show that all the positive strand RNA viruses tested produced copious amounts of dsRNA in infected cells, this antibody has been used extensively in a wide range of systems, as documented in over 200 scientific publications. J2 can be used to detect dsRNA intermediates of viruses as diverse as Hepatitis C virus, Dengue virus,rhinovirus, Chikungunya virus, Rabies virus, Polio virus, Classic swine fever virus, Brome mosaic virus and many more in cultured cells and also in fixed paraffin-embedded histological samples. J2 has been used to elucidate how anti-viral responses are initiated, what counter-strategies viruses have adopted to avoid them,and to explore the viral life cylce by enabling ultrastructiural localisation studies of viral nucleic acid replication sites (Welsch et al., 2009 & Knoops et al., 2011). J2 has also been recommended as a diagnostic tool to detect whether an unkown pathogen is bacterial or viral in nature (Richardson et al., 2010).Recently J2 has also been used to monitor the removal of dsRNA from in vitro synthethised mRNA preparations that may have potential use in gene therapy (Kariko et al., 2011). J2 has been used successfully in various immunocapture methods, such as ELISA.

Intended Use:

The KRIBIOLISA™ Double-Stranded RNA (dsRNA) ELISA is used as an analytical tool for the qualitative /quantitative determination of Double-Stranded RNA (dsRNA) in mRNA based preparations. We recommend using the ELISA 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 quantitative control.

Note: 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 J2 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 quantitative 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 absorbance values at 450nm.

Principle:

The KRIBIOLISA™ Double-Stranded RNA (dsRNA) ELISA employs the quantitative sandwich enzyme immunoassay technique. It is based on the use of two double-stranded RNA (dsRNA)-specific monoclonal antibodies which allows sensitive and selective detection of dsRNA molecules (>=40 bp), independent of their nucleotide composition and sequence.Antibodies to dsRNA (J2) are pre-coated onto microwells. Samples and standards are pipetted into microwells and are bound by the capture antibody. Then, a HRP (horseradish peroxidase) conjugated Anti-dsRNA (K1) is pipetted and incubated. After washing microwells in order to remove any non-specific binding, the ready to use substrate solution (TMB) is added to microwells and color develops proportionally to the amount of dsRNA in the sample. Color development is then stopped by addition of stop solution. Absorbance is measured at 450 nm.