Double-stranded RNA (dsRNA) ELISA kit (J2 based)

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 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 ?l and 200 ?l
? Multichannel pipettes ? 200 ?l

? 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 4 ?l RNase-free MilliQ water. The concentration will be 1 ?g/?l 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 01 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 30 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 ?l 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 ?l washing solution/well. Do not allow wells to dry before adding the next solution.
8. Pipette 100 ?l 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 1.4 ?l 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 ?l 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 ?l 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 ?l of 2M H2SO4 to all wells. Read absorbance at 450 nm blanking on the Zero Standard.

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 J2 (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 J2 anti-dsRNA IgG2a monoclonal antibody 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.

Infectious Agents & Disease|Microbiology

Double-stranded RNA (dsRNA) ELISA kit containing the following reagents to perform 500 tests (5x96 well plates):
? Reagent 01: 1 vial of coating antibody (store at -20 °C)
? Reagent A: 1 vial of Double stranded RNA control Poly(I:C) (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

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 J2 and K2.

dsRNA ELISA kit