AffiGEN® double-stranded RNA (dsRNA) ELISA Assay Kit

AffiGEN® double-stranded RNA (dsRNA) ELISA Assay Kit

The AffiGEN® dsRNA ELISA Kit is designed to detect and measure double-stranded RNA (dsRNA) contamination in in vitro transcribed (IVT) mRNA products. dsRNA is a common byproduct formed during mRNA production, and its presence can trigger immune responses and reduce the effectiveness of mRNA-based vaccines and therapies. Removing or minimizing dsRNA is essential for developing safe and high-quality mRNA drugs.

This ELISA kit uses a highly specific anti-dsRNA antibody that recognizes only double-stranded RNA, without cross-reacting with single-stranded RNA or DNA. It provides reliable, sensitive, and quantitative results, making it useful across different stages of mRNA drug development—from early research and process optimization to final product quality control.

Intended use:

This kit is designed for the quantitative measurement of residual double-stranded RNA (dsRNA).

Storage conditions:

• Store the entire kit at 2–8°C for the duration of its shelf life. To maintain stability, avoid exposure to strong light.
• After opening the microplate, cover any unused wells with the plate sealer and place it back into the original foil pouch with the desiccant pack. Seal the pouch tightly and return it to 2–8°C as soon as possible.
• All other reagents should also be returned to 2–8°C immediately after use to preserve performance.

Kit components:

Materials Required But Not Supplied:

• Microplate reader with a 450 ± 10 nm filter (preferably with detection capability at both 450 nm and 650 nm wavelengths).
• Microplate shaker.
• RNase-free tips and centrifuge tubes.

Procedure workflow:

1. Prepare reagents

• Get all necessary reagents ready (standards, samples, buffers, DA conjugate, HRP-SA, TMB substrate, stop solution).

2. Add standards and samples – 100 µL/well

• Pipette 100 µL of both standards and unknown samples into each well of the ELISA plate.

3. Incubate

• At room temperature (R.T.)
• 500 rpm shaking
• For 60 minutes

4. Discard liquid and snap plate on absorbent paper

• Invert the plate to remove contents.
• Tap the plate firmly on absorbent paper to remove residual fluid.

5. Wash with 1× wash buffer – 4 times

• Wash each well with the buffer.
• Typically involves filling, soaking briefly, and discarding 4 times.

6. Add biotinylated DA – 100 µL/well

• Dispense 100 µL of biotinylated detection antibody (DA) into each well.

7. Incubate

• R.T.
• 500 rpm shaking
• For 60 minutes

8. Discard liquid and snap plate on absorbent paper

• Remove unbound detection antibody.

9. Wash with 1× wash buffer – 4 times

• Same washing procedure to remove any excess unbound detection antibody.

10. Add HRP-SA – 100 µL/well

• Add 100 µL of horseradish peroxidase-streptavidin (HRP-SA) conjugate to each well.

11. Incubate

• R.T.
• 500 rpm shaking
• For 30 minutes

12. Discard liquid and snap plate on absorbent paper

• Remove unbound HRP-SA.

13. Wash with 1× wash buffer – 4 times

• Final wash to ensure minimal background noise in detection.

14. Add TMB substrate – 100 µL/well

• Add 100 µL of tetramethylbenzidine (TMB) solution.
• This reacts with HRP to produce a blue color.

15. Incubate at R.T. in the dark for 30 minutes

• Allow color to develop.
• Protect from light to avoid degradation of the TMB.

16. Add stop solution – 50 µL/well

• Typically sulfuric acid (H₂SO₄) or another acidic solution to stop the reaction.
• The color changes from blue to yellow.

17. Read absorbance at OD450/650 nm

• Measure the absorbance using a microplate reader.
• OD450 is for quantification; OD650 may be used as reference.