Double-stranded RNA (dsRNA) ELISA kit (K1 based)

Synonyms :dsRNA ELISA kit

Isotype : IgG2a kappa/IgM kappa

Fieldofinterst : Infectious Agents & Disease|Microbiology

Purificationmethod :

Concentration :

Applications Description : 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:
?5 ELISA plates (96 well)
? Microtiter plate reader spectrophotometer with dual wavelength capability at 370 and 450 nm.
? Single channel pipettes ¡V 10 ul and 200 ul
? Multichannel pipettes ¡V 200 ul
? 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 ¢XC.
? 2 M H2SO4

Preparation of Reagents and ELISA Plates
? Reconstitute Reagent A by adding 20 ul RNase-free MilliQ water. The concentration will be 1 ug/ul dsRNA. (Store it at -20 ¢XC or -80 ¢XC.)
? 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 ƒÝl/well in 2 ELISA plates.
? Cover the plates and incubate them overnight at 4 „aC.
? Discard contents of wells into waste. Add 100 ƒÝl/well 1% BSA in PBS + 0.2% NaN3 to each well and incubate at 37 „aC 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 ƒÝl/well PBS containing 0.2% sodium azide.
Wrap plates in plastic foil and store them refrigerated at 4 „aC. 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 ul antigen to duplicated wells in the plate.
6. Cover and incubate for 60 min at 37 ¢XC.
7. Discard contents of wells into waste. Wash plate 4 times with PBS + 0.5 % Tween 20 adding 250 ul washing solution/well. Do not allow wells to dry before adding the next solution.
8. Pipette 100 ul undiluted Reagent B into all wells.
9. Cover and incubate for 60 minutes at 37 ¢XC.
10. During the incubation (step 9) dilute Reagent C by pipetting 1.6 ul 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 ul diluted Reagent C to each well.
13. Cover and incubate for 60 minutes at 37 ¢XC.
14. Wash as in Step 7. Take care to remove all washing fluid after the last wash.
15. Pipette 100 ul undiluted Reagent D into each well.
16. Cover and incubate in the dark at room temperature (20¡Ó5 ¢XC).
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 ul of 2M H2SO4 to all wells. Read absorbance at 450 nm blanking on the Zero Standard.

Storage: at -20ºC