dsRNA ELISA Kits

Calibration curve range	UTP dsRNA : 7.7 – 1,000 pg/mL m1ψ dsRNA : 15.4 – 2,000 pg/mL
Assay target	dsRNA (double-stranded RNA)
Analysis sample	mRNA solution
Measurement duration	Approx. 4.5 hours
Detection method	Colorimetric assay
Wavelength	Primary wavelength 450 nm Secondary wavelength 620 nm

Example of Calibration Curve

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This kit detects dsRNA by sandwich ELISA.
Each well of the assay plate is coated with anti-dsRNA antibody clone J2, and the standard solution or sample is added to the wells.
Biotin-labeled anti-dsRNA antibody clone J2 and peroxidase-conjugated streptavidin are then added and allowed to react.
Finally, the peroxidase remaining in the wells is reacted with TMB solution, enabling quantitative measurement of dsRNA by colorimetric detection.

Kit Contents

Components	Amount
Antibody-coated Plate	96 wells (8×12) /1 plate
UTP dsRNA Standard	50 μL/1 bottle
m1ψ dsRNA Standard	50 μL/1 bottle
Sample Reaction Buffer	25 mL/1 bottle
Antibody Reaction Buffer	25 mL/1 bottle
Biotin-conjugated Antibody	1 bottle
Peroxidase-conjugated Streptavidin Solution	120 μL/1 bottle
TMB Solution	12 mL/1 bottle
Stop Solution	12 mL/1 bottle
Wash Solution (10×)	100 mL/1 bottle
Plate Seal	4 sheets

Protocol

Step 01

Reagent preparation
Dilute the standards and wash solution included in the kit.

Step 02

Sample preparation
Dilute samples as required.

For UTP mRNA samples, we recommend adjusting the concentration to 100 ng/mL mRNA or less. For m1ψ mRNA samples, a concentration of 500 ng/mL mRNA or less is recommended. Adjust the sample concentrations as necessary to ensure they fall within the calibration curve range.

Step 03

Reaction procedure

① Antibody-coated plate	Wash 3 times
② Sample or standard solution	Add 100 μL Approximately shake at 500 rpm, 20℃ - 25℃, incubate for 2 hours *Preparation of biotin-conjugated antibody solution from the supplied antibody (Dissolve in 120 μL of purified water, then dilute 100-fold with antibody reaction buffer equilibrated to room temperature.)
③ Antibody-coated plate	Wash 3 times
④ Biotin-conjugated antibody Solution	Add 100 μL Approximately shake at 500 rpm, 20℃ - 25℃, incubate for 1 hour
⑤ Antibody-coated plate	Wash 3 times
⑥ Peroxidase-conjugated streptavidin solution	Add 100 μL Approximately shake at 500 rpm, 20℃ - 25℃, incubate for 1 hour
⑦ Antibody-coated plate	Wash 3 times
⑧ TMB solution	Add 100 μL Approximately shake at 600 rpm for 10 seconds, repeated 3 times, then let stand, 20℃ - 25℃, Incubate for 30 minutes
⑨ Stop solution	Add 100 μL Approximately shake at 600 rpm for 10 seconds, repeated 3 times

Step 04

Absorbance measurement
Measure absorbance with a plate reader at a primary wavelength of 450 nm and a secondary wavelength of 620 nm.

Effect of Nucleotide Modification and Chain Length

capture : K1 detection : K2

capture : J2 detection : K2

capture : J2 detection : J2

The detection system of this product (capture: J2; detection: biotinylated J2) was compared with combinations of other anti-dsRNA-recognizing antibody clones. UTP and m1ψ dsRNA standards were used to measure dsRNA of various chain lengths at a concentration of 1 ng/mL.
The results demonstrated that the detection system of this product exhibited the least variation in detected concentration, regardless of m1ψ modification or chain length.

Measurement of dsRNA in mRNA Solutions

Using this kit, the dsRNA concentrations in capped unmodified and m1ψ-modified mRNA solutions were measured.
The measured values were proportional to the mRNA sample concentration and showed linearity over the ranges of 20 to 100 ng/mL for unmodified mRNA and 50 to 500 ng/mL for m1ψ-modified mRNA.

Spike and Recovery Test

UTP mRNA

Specimen	Spiked amount of dsRNA (pg/mL)	Measurement value of dsRNA (pg/mL)	Recovery (%)
mRNA solutions 200 ng/mL	-	176	-
	20	197	104
	100	285	109
	500	637	92
mRNA solutions 50 ng/mL	-	44	-
	20	64	100
	100	140	96.5
	500	550	101

m1ψ mRNA

Specimen	Spiked amount of dsRNA (pg/mL)	Measurement value of dsRNA (pg/mL)	Recovery (%)
mRNA solutions 200 ng/mL	-	167	-
	20	186	93
	100	280	113
	500	638	94.1
mRNA solutions 50 ng/mL	-	41.1	-
	20	60	95.7
	100	145	104
	500	521	95.9

Using this kit, a spike-and-recovery test for dsRNA in mRNA solutions was performed.
The results showed high recovery of dsRNA at mRNA concentrations of 50 ng/mL and 200 ng/mL.

Measurement of dsRNA in IVT Solutions after IVT Reaction

Using two different commercially available in vitro transcription (IVT) kits (A and B), two types of mRNA, one long and one short, were synthesized.
Using this product, the dsRNA concentrations in synthesized mRNA solutions were measured.
The measured values were confirmed to be linear with respect to the mRNA sample concentration over the range of 25 to 100 ng/mL for unmodified mRNA and 125 to 500 ng/mL for m1ψ modified mRNA.

Matrix Effect of IVT Reaction Buffer

A comparison was conducted between this kit and another manufacturer’s kit to evaluate the recovery of dsRNA spiked into the IVT reaction mixture prior to synthesis.
Unlike the other manufacturer's product, this kit was not affected by the matrix and was able to measure dsRNA accurately without requiring sample dilution.