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dsRNA ELISA Kit

dsRNA ELISA Kit

Catalog Number: abs590014 Brand: Absin
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$1,250.00
$1,250.00
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Product Details

Product Specification

Usage

Ⅰ. Required instruments and reagents:
1. Microplate reader (including 450nm wavelength, it is recommended to include dual wavelength detection mode, the main and auxiliary wavelengths are 450nm and 650nm respectively).
2. Microplate shaker.
3. RNase-free gun tip and EP tube.

Ⅱ. Preparation before the experiment:
1. Equilibrate the reagents used in this experiment to room temperature (18-25 ℃)
2. 20 × concentrated washing liquid is diluted with purified water in a volume ratio of 1:19 to form a washing working liquid.
3. Centrifuge the antibody tube, HRP-SA tube and standard tube at 1000 rpm for 30s before use to avoid residual reagents on the tube wall and cover.
4. 100 × biotinylated detection antibody and 100 × HRP-streptavidin were diluted 100 times with diluent before use and then used.
5. Unmodified, pUTP modified dsRNA standards were diluted to 1, 0.5, 0.25, 0.125, 0.0625, 0.0312, 0.0156, 0 pg/μL with STE buffer. N1-Me-pUTP modified dsRNA standards were diluted to 2, 1, 0.5, 0.25, 0.125, 0.0625, 0.0312, 0 pg/μL with STE buffer. 5-OMe-UTP modified dsRNA standards were diluted to 4, 2, 1, 0.5, 0.25, 0.125, 0.0625, 0 pg/μL with STE buffer.

Dilution methods are recommended as follows:
(1) No modification, pUTP modification

serial number Final Concentration (pg/μL) Dilution Method
STE buffer Working Standard
  100 49 μL 1 μL 5ng/μL standard
A 1 495 μL 5 μL of 100pg/μL solution
B 0.5 250 μL 250 μL A solution
C 0.25 250 μL 250 μL B solution
D 0.125 250 μL 250 μL C solution
E 0.0625 250 μL 250 μL D solution
F 0.0312 250 μL 250 μL E solution
G 0.0156 250 μL 250 μL of F solution
H 0 250 μL /

(2) N1-Me-pUTP Modification
serial number Final Concentration (pg/μL) Dilution Method
STE buffer Working Standard
  100 49 μL 1 μL 5ng/μL standard
A 2 490 μL 10 μL of 100pg/μL solution
B 1 250 μL 250 μL A solution
C 0.5 250 μL 250 μL B solution
D 0.25 250 μL 250 μL C solution
E 0.125 250 μL 250 μL D solution
F 0.0625 250 μL 250 μL E solution
G 0.0312 250 μL 250 μL of F solution
H 0 250 μL /

(3) 5-OMe-UTP Modification
serial number Final Concentration (pg/μL) Dilution Method
STE buffer Working Standard
  100 49 μL 1 μL 5ng/μL standard
A 4 480 μL 20 μL of 100pg/μL solution
B 2 250 μL 250 μL A solution
C 1 250 μL 250 μL B solution
D 0.5 250 μL 250 μL C solution
E 0.25 250 μL 250 μL D solution
F 0.125 250 μL 250 μL E solution
G 0.0625 250 μL 250 μL of F solution
H 0 250 μL /

Ⅲ. Experimental steps:
1. Take out the required slats from the aluminum foil bag after room temperature equilibrium, cover the remaining slats with a sealing film, seal them with a ziplock bag and put them back at 2 ~ 8 ℃.
2. Set standard wells and sample wells, add 100μL of standard substances of different concentrations to each standard well, and add 100μL of sample to be tested to the sample well.
Note: When the dsRNA content in the sample to be tested cannot be determined, the STE buffer should be used to make multiple dilution factors for detection, so as to avoid the content being too high and the valid value cannot be read.
3. Seal the reaction holes with a plate sealing membrane, and vibrate the plate (500rpm) for 60 minutes at room temperature.
4. Discard the liquid, pat dry on absorbent paper, fill each well with washing liquid (250μL), let stand for 30s, throw away the washing liquid, pat dry on absorbent paper, and repeat washing 4 times.
5. Add 100μL of biotinylated detection antibody at working concentration to each well of the standard well and sample well, seal the reaction well with a plate sealing membrane, and vibrate the plate (500rpm) at room temperature for 60 minutes.
6. Discard the liquid, pat dry on absorbent paper, fill each well with washing liquid (250μL), let it stand for 30s, throw away the washing liquid, pat dry on absorbent paper, and repeat washing 4 times.
7. Add 100μL of HRP-streptavidin with working concentration to each well of the standard well and sample well, seal the reaction well with a plate sealing membrane, and vibrate the plate (500rpm) at room temperature for 30 minutes.
8. Discard the liquid, pat dry on absorbent paper, fill each well with washing liquid (250μL), let stand for 30s, throw away the washing liquid, pat dry on absorbent paper, and repeat washing 4 times.
9. Add 100μL of one-component substrate chromogenic solution to each well, seal the reaction well with a sealing film, and let it stand at room temperature to protect from light for 30 minutes.
10. Add 50μL of stop solution to each well, detect immediately, and set the wavelength of the microplate reader at 450nm (dual wavelength 450nm/650nm is recommended).

Ⅳ. Interpretation of test results:
1. Take the average light absorption value of the standard product, the blank control, and the sample, and subtract the average light absorption value of the blank control to obtain the light absorption calibration value of the standard product and the sample. The standard curve is drawn with the standard concentration as the abscissa and the calibrated standard light absorption value as the ordinate. (The light absorption value in the dual-wavelength detection mode is 450 nm minus 650 nm)
2. It is recommended to use professional curve making software, such as curve expert 1.3 or ELISA Calc (please use Logistic five-parameter or four-parameter fitting curve calculation method to draw standard curves), etc.

Ⅴ. Detection performance
1. Detection limit:
No modification, pUTP modification, N1-Me-pUTP modified dsRNA detection limit ≤ 0. 001 pg/μL, 5-OMe-UTP modified dsRNA detection limit ≤ 0. 01 pg/μL.
2. Limit of quantification:
No modification, pUTP-modified dsRNA limit of quantification was 0.0156 pg/μL, N1-Me-pUTP-modified dsRNA limit of quantification was 0.0312 pg/μL, and 5-OMe-UTP-modified dsRNA limit of quantification was 0.0625 pg/μL.
3. Precision:
Intra-plate coefficient of variation ≤ 10%, inter-plate coefficient of variation ≤ 10%
4. Recovery rate:
80%~120%
5. Linear range:
The linear range of unmodified, pUTP-modified dsRNA detection was 0.0156-0.5 pg/μL, the linear range of N1-Me-pUTP-modified dsRNA detection was 0.0312-1 pg/μL, and the linear range of 5-OMe-UTP-modified dsRNA detection was 0.0625-1 pg/μL.
Theory This kit adopts the principle of double-antibody sandwich method and is coupled with biotin-streptavidin system to quantitatively detect the content of double-stranded RNA (dsRNA) in samples. The length of the detected dsRNA is 60 bp or more, and the detected dsRNA is independent of its nucleic acid sequence. The microwells of the plate were coated with anti-dsRNA antibody, incubated and washed, and then incubated with biotinylated detection antibody to form antibody-antigen-antibody complex. After washing again, horseradish peroxidase (HRP) labeled streptavidin (SA) was added. After thorough washing, the substrate TMB was added to develop color, and TMB was converted to blue under the catalysis of peroxidase and to final yellow by acid termination.
Synonym Double-stranded RNA ELISA test kit
Composition
Serial number Name Specifications
1 Reaction plate 8 × 12 wells
2 Biotinylated detection antibody (100 ×) 120 μL
3 HRP-Streptavidin (100 ×) 120 μL
4 Diluent 30mL
5 Chromogenic liquid 12mL
6 Stop liquid 6mL
7 Concentrated wash (20 ×) 40mL
8 dsRNA standard (no modification, 5ng/μL) 15 μL
9 dsRNA standard (pUTP modified, 5ng/μL) 15 μL
10 dsRNA standard (N1-Me-pUTP modified, 5ng/μL) 15 μL
11 dsRNA standard (5-OMe-UTP modification, 5ng/μL) 15 μL
12 STE buffer 50mL
13 Sealing film 4 sheets
General Notes 1. The color development temperature and time are crucial to the experimental results and should be accurately grasped.  
2. During the washing process, the washing liquid should be soaked in the reaction plate for 30 seconds and then dried to fully wash the non-specifically adsorbed components.  
3. All reagents should be thoroughly shaken before use. When adding samples, the added sample should be added to the bottom of the well of the enzyme label plate to avoid adding it to the upper part of the well wall. When adding samples, be careful not to spill or produce bubbles.  
4. If crystals are found in the concentrated washing liquid, they can be incubated in a 37 °C water bath, and then mixed and diluted to the working concentration after the crystals are completely dissolved.  
5. Avoid introducing sodium azide (NaN) into the sample3), sodium azide will destroy horseradish peroxidase activity and make the detection value low.  
6. RNase contamination should be strictly avoided during the experimental operation.  
7. Do not use a shaker instead of a vibrating plate machine. If there is no vibrating plate machine, room temperature incubation can be used, but standing incubation will cause the detection sensitivity to be about doubled.  
It is recommended that unmodified, pUTP modified standards be diluted from 2pg/μL, N1-Me-pUTP modified standards be diluted from 4pg/μL, 5-OMe-UTP modified standards be diluted from 8pg/μL, and the HRP-SA incubation duration be adjusted from 30min to 60min.
Storage Temp. 1. The kit is stored at 2 ~ 8 °C, avoid direct strong light, and is valid for 12 months.
2. After the coating strip is unpacked and used, the remaining coating strip should be sealed, stored at 2 ~ 8 °C, and used within the validity period.
3. Other components of the kit should be put back to 2 ~ 8 ℃ in time after use and used within the validity period.

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