| Product Description |
Double-stranded RNA
(dsRNA) is a by-product of the in vitro transcription (IVT) of
mRNA and can be co-eluted with mRNA by poly-dT affinity chromatography. The dsRNA
significantly increases the immune response to mRNA vaccines, causing inflammation
in recipients of the treatment. Therefore, it is a notable challenge for mRNA vaccine manufacturers. The removal and detection of dsRNA is significant
for mRNA in vitro transcription (IVT) processes. Anti-dsRNA monoclonal antibody
(J2), which sensitively and selectively recognizes dsRNA structure (larger than
30-40 bp) in mRNA molecules, independent of their nucleotide composition and
sequence, is widely used to assess the dsRNA amounts in mRNA. The J2 antibody can also be used to detect dsRNA
in self-amplifying RNAs (saRNAs) containing the modified nucleotides such as
5-methylcytidine (m5C), 5-methyluridine (m5U) and N1-methylpseudouridine (m1Ψ) by immuno-dot blot[1]. The dsRNA-specific
antibody (J2) has been broadly widely used to detect dsRNA in over 200
scientific publications[2-8].
|
| LLOQ |
0.123 ng/mL
|
| ULOQ |
30 ng/mL
|
| Precision |
Intra-assay: CV≤10%
Inter-assay: CV≤15%
|
| Specificity |
dsRNA with nature nucleotides and dsRNA containing
modified nucleotides
|
| Kit Contents |
| Component |
Quantity/Size |
Part No. |
| Capture Plate |
1 plate |
T1-80 |
| Standard Stock |
1 vial (25 μL) |
T1-10 |
| 100×Detection Antibody |
2 vials (Lyophilized) |
T1-20 |
| 10×HRP-Conjugated Antibody |
1 vial (1.50 mL) |
T1-30 |
| Sample Dilution Buffer |
1 bottle (50 mL) |
T1-60 |
| Detection Antibody Dilution Buffer |
1 bottle (12 mL) |
T1-61 |
| HRP-Conjugated Antibody Dilution Buffer |
1 bottle (12 mL) |
T1-62 |
| 20×Wash Solution |
1 bottle (60 mL) |
T1-70 |
| TMB Solution |
1 bottle (12 mL) |
A1-40 |
| Stop Solution |
1 bottle (6 mL) |
A1-50 |
| Plate Sealer |
2 pieces |
N/A |
| User Manual |
1 piece |
N/A |
|
| Storage |
The
unopened kit is stable for at least 12 months from the date of manufacture at 2-8°C,
and the opened kit is stable for up to 30 days from the date of opening at 2-8°C. |
| Assay Principle |
This dsRNA
Detection Kit can be used for the analysis of dsRNA amounts in mRNA samples. It
is a sandwich ELISA assay with two kinds of the J2 antibodies. One of J2
antibody is used as a capture antibody to bind dsRNA on the plate, another J2 antibody is used as the detection
antibody. A 400-bp dsRNA standard with N1-methylpseudouridine (m1Ψ) is used
in the kit. When standards or samples are added, dsRNA can be bound by the
capture antibody on the plate. Then, another J2 antibody (the detection
antibody) is added to the plate, and HRP-Conjugated Antibody, only recognizing
the detection antibody, is added. Finally, TMB solution is added to the plate
and color development is stopped by addition of stop solution. The color
intensity can be read at 450 nm by a microplate reader. The quantity of dsRNA
in the sample is quantified against a dsRNA standard curve.
For accurate dectection
of dsRNA residual in the samples, the dsRNA standards with different sequence,
length and modified nucleotides including m5C, m5U or m1Ψ, should be taken into
consideration. GenScript can synthesise dsRNAs of various sequences according
to the needs of customers, which can be used as reference standards for the
detection of dsRNAs. |
| Reference |
1.
Hiva Azizi et al. (2024) Self-amplifying RNAs generated with the modified nucleotides
5-methylcytidine and 5-methyluridine mediate strong expression and
immunogenicity in vivo. NAR Molecular Medicine.
2.
Schönborn et al. (1991) Monoclonal antibodies to double-stranded
RNA as probes of RNA structure in crude nucleic acid extracts. Nucleic Acids
Res. PMID: 2057357
3.
Maria Bokarewa et al.
(2008) Arthritogenic dsRNA is present in synovial fluid from rheumatoid
arthritis patients with an erosive disease course. Eur J Immunol. Viruses.
PMID: 31615058
4.
Ruikang Liu et al. (2016)
Opposing Roles of Double-Stranded RNA Effector Pathways and Viral Defense
Proteins Revealed with CRISPR-Cas9 Knockout Cell Lines and Vaccinia Virus
Mutants. J Virol. PMID: 27334583
5.
Markus Baiersdörfer et
al. (2019) A Facile Method for the Removal of dsRNA Contaminant from In
Vitro-Transcribed mRNA. Mol Ther Nucleic Acids. PMID: 30933724
6.
Ashish Dhir et al. (2018)
Mitochondrial double-stranded RNA triggers antiviral signalling in humans.
Nature. PMID: 30046113
7.
Yimeng Gao et al. (2021)
Transcriptome-wide quantification of double-stranded RNAs in live mouse tissues
by dsRIP-Seq. STAR Protoc. PMID: 33778776
8.
Carolyn J. Decker et al.
(2019) dsRNA-Seq: Identification of viral infection by purifying and sequencing
dsRNA. PMID: 31615058 |
Figure 1: Double-stranded RNA(dsRNA) ELISA Kit
For research use only.
Not intended for human and animal therapeutic or diagnostic use.
