Assay Protocol Summary
- Bring all components to room temperature (18–25°C); use RNase-free consumables throughout
- Prepare Poly(I:C) standards: reconstitute lyophilised vial in 1 mL Sample Diluent (1,000 ng/mL), dilute to 200 ng/mL, then serial dilute (200 → 100 → 50 → 25 → 12.5 → 6.25 → 3.125 → 0 ng/mL)
- Add 100 µL standards or diluted samples to respective wells
- Seal plate; incubate 120 minutes at 37°C
- Wash 4× with Wash Buffer (1X)
- Add 100 µL K1 Detection:HRP Conjugate to all wells
- Seal plate; incubate 60 minutes at 37°C
- Wash 4× with Wash Buffer (1X)
- Add 100 µL TMB substrate; incubate 30 minutes at room temperature in dark (wells turn blue)
- Add 100 µL stop solution (blue → yellow)
- Read absorbance at 450 nm within 30 minutes
Frequently Asked Questions
1. What does the J2 antibody detect?
The J2 monoclonal antibody (IgG2a kappa) specifically binds double-stranded RNA molecules ≥40 base pairs in length. Detection is sequence-independent — the antibody recognises the A-form double-helical structure of dsRNA regardless of nucleotide composition. All naturally occurring dsRNA species investigated to date (40–50 species) are recognised, as well as synthetic analogs including poly(I:C) and poly(A):poly(U).
2. Can this kit detect dsRNA in mRNA preparations after IVT purification?
Yes. This is one of the primary designed applications. The kit is sensitive enough (LOD 0.3 ng/mL) to detect trace dsRNA remaining after HPLC or cellulose-based purification of IVT mRNA. The quantitative mode allows you to measure the effectiveness of each purification step and confirm specification limits are met. Dilute samples appropriately to bring residual dsRNA within the 3.125–200 ng/mL working range.
3. Will the kit detect mRNA itself?
No. Single-stranded mRNA is not detected by the J2 or K1 antibodies. The assay is highly selective for double-stranded RNA ≥40 bp and shows negligible reactivity with ssRNA, ssDNA, dsDNA, tRNA, siRNA, or short RNA duplexes below the 40 bp threshold.
4. What standard is used for quantification?
The kit includes lyophilised Poly(I:C) [polyinosinic:polycytidylic acid] as the dsRNA reference standard, reconstituted at 1,000 ng/mL. Poly(I:C) is a synthetic dsRNA analog widely used as a TLR3 agonist and immunostimulatory research tool. Note that J2’s affinity for poly(I:C) is approximately 10-fold lower than for some natural dsRNA antigens — quantification results for natural viral dsRNAs are expressed as ng/mL poly(I:C) equivalents.
5. What sample types are compatible?
The kit has been validated in mRNA-based preparations, nucleic acid extracts, human serum, human plasma, lysates, and viral culture supernatants. Matrix effect studies showed minimal interference in serum and plasma. Use RNase-free conditions for RNA-containing samples.
6. Can the kit detect dsRNA from specific viruses?
Yes — the J2 antibody has been used to detect dsRNA from Hepatitis C virus, Dengue virus, Chikungunya virus, Rhinovirus, Rabies virus, Poliovirus, Classic swine fever virus, Brome mosaic virus, and many others. The sequence-independent detection means any dsRNA replication intermediate ≥40 bp will be captured regardless of the virus species.
7. Is this a qualitative or quantitative assay?
Both. The kit supports qualitative screening (positive/negative relative to a positive control) and quantitative measurement (ng/mL from the standard curve). The format accommodates both lot release go/no-go testing and process characterisation workflows requiring actual concentration values.
8. What is the High Dose Hook Effect and how do I avoid it?
At very high dsRNA concentrations — most likely in samples from early in the purification process before dsRNA removal — the signal may appear lower than a diluted sample, indicating antibody saturation. If your undiluted sample reads lower than a 1:2 or 1:4 dilution, dilute further until results are consistent with the dilution factor. The kit has been validated to 1:32 dilution.
Downloads & Resources
- Validation Guide (FDA/ICH ICH Q2(R1)) — Full validation data: sensitivity, linearity, precision, parallelism, matrix effect, specificity. Download PDF →
- Product Datasheet / Kit Insert (Ver 1.3) — Full assay protocol, standard preparation, kit contents. Download PDF →
