dsRNA (J2 based) ELISA
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 cycle by enabling ultrastructural 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 unknown 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 synthetized 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.
Krishgen’s robust and sensitive dsRNA ELISA kit allows qualitative / semi-quantitative screening of double stranded RNA 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.
Krishgen’s dsRNA is one of the only commercially available ELISA for the quantitative measurement of dsRNA. The ELISA kit comes with a lyophilized standard for running calibration. Additionally, if you prefer to have the kit include Poly I:C positive controls, Krishgen has an option for that as well.
Krishgen understands that time and effort is key for running contamination detection tests. To reduce time of the assay, Krishgen comes with pre-coated plates that are pre-optimized and validated for detection of dsRNA. No other kit currently comes with pre-coated plates, which means researchers spend over 24 hours simply coating and blocking the plates. Coating manually also increases chance of error and requires additional validation and optimization before running the kit.
Krishgen’s kits are ready to use – simply dilute the lyophilized standards, prep your samples, and run the kit like any other ELISA.
This kit has been validated as per EMA/FDA guidelines in line with ICH Code for Harmonization of Biological Assays and the Assay Guidance Manual. Thorough validation includes testing for sensitivity, reproducibility, and intra- / inter- assay precision. This dsRNA ELISA clocks in at an LOD of 1.5 ng/ml.
Precision:
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.
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