Human high-sensitivity C-reactive protein
(
hs-CRP
)
ELISA
Reagent
Box operation instructions
This kit is for research use only.
Experimental principle
The kit uses a double antibody sandwich method to determine human high-sensitivity C-reactive protein in specimens.
(
hs-CRP
)
Level. Purified human high-sensitivity C-reactive protein
(
hs-CRP
)
The antibody is coated with a microplate to prepare a solid phase antibody, and high-sensitivity C-reactive protein is sequentially added to the microwell of the coated monoclonal antibody.
(
hs-CRP
)
HRP-labeled high-sensitivity C-reactive protein
(
hs-CRP
)
The antibody binds to form an antibody-antigen-enzyme-labeled antibody complex, which is thoroughly washed and then added to the substrate TMB for color development. TMB is converted to blue under the catalysis of HRP enzyme and converted to the final yellow color by the action of an acid. Color depth and high-sensitivity C-reactive protein in the sample
(
hs-CRP
)
Positive correlation. Determination of absorbance at 450 nm using a microplate reader
(
OD value
)
Calculate human high-sensitivity C-reactive protein in the sample by standard curve
(
hs-CRP
)
concentration.
Human high-sensitivity C-reactive protein
(
hs-CRP
)
ELISA
Reagent
box
Steps
1. Dilution of standard: This kit provides one original standard, which can be diluted in a small tube according to the following chart.
Add 40μl standard dilution of 40μg/L No.5 standard 150μl standard
Add 20 μg/L No. 4 standard 150 μl of No. 5 standard to 150 μl of standard dilution
Add 10 μg/L No. 3 standard 150 μl of No. 4 standard to 150 μl of standard dilution
Add 5 μg/L No. 2 standard 150 μl of No. 3 standard to 150 μl of standard dilution
Add 2.5 μg/L No. 1 Standard 150 μl Standard No. 2 to 150 μl Standard Diluent
2. Sample loading: separate blank holes
(
Blank control wells are not added with samples and enzyme-labeled reagents, and the remaining steps are the same.
)
, standard hole, sample hole to be tested. Accurately load 50μl of the standard on the enzyme-labeled plate, add 40μl of the sample dilution to the well to be tested, and then add 10μl of the sample to be tested.
(
The final dilution of the sample is 5 times
)
. Add the sample to the bottom of the well of the microplate, try not to touch the wall of the well, and shake gently to mix.
3. Incubation: The plate was sealed with a sealing film and incubated at 37 ° C for 30 minutes.
4. Dosing: 30 times concentrated washing solution diluted with distilled water 30 times and used
5. Washing: Carefully remove the sealing film, discard the liquid, dry it, fill each well with the washing liquid, let stand for 30 seconds, then discard it, repeat 5 times, and pat dry.
6. Add enzyme: 50 μl of enzyme labeling reagent was added to each well, except for blank wells.
7. Incubation: The operation is the same as 3.
8. Washing: The operation is the same as 5.
9. Color development: Add 50 μl of the developer to each well, then add 50 μl of the developer, gently shake and mix, and develop at 37 ° C for 10 minutes.
10. Termination: 50 μl of stop solution per well, stop the reaction
(
At this time, the blue turns yellow.
)
.
11. Determination: The absorbance of each well was measured sequentially with blank air conditioner zero and 450 nm wavelength.
(
OD value
)
. The measurement should be carried out within 15 minutes after the addition of the stop solution.
Human high-sensitivity C-reactive protein
(
hs-CRP
)
ELISA
Reagent
box
Precautions
1. The kit should be taken out from the refrigerated environment and should be used at room temperature for 15-30 minutes before use. If the enzyme label is unsealed after opening, the slats should be stored in a sealed bag.
2. Concentrated washing liquid may crystallize out. When diluted, it can be heated and dissolved in a water bath. The washing will not affect the result.
3. The sampler should be used for each step, and the accuracy should be corrected frequently to avoid test errors. It is best to control the loading time within 5 minutes. If the number of specimens is large, it is recommended to use a gun.
4. Please make a standard curve at the same time of each measurement, it is best to make a double hole. If the content of the substance to be tested in the specimen is too high
(
The OD value of the sample is larger than the OD value of the first hole of the standard product hole.
)
, please first dilute a certain multiple with the sample diluent
(
n times
)
After the measurement, please multiply the total dilution factor by the end of the calculation.
(
×n×5
)
.
5. The sealing film is intended for single use only to avoid cross-contamination.
6. Please keep the substrate away from light.
7. strictly follow
Human high-sensitivity C-reactive protein
(
hs-CRP
)
ELISA
Reagent
box
The operation of the manual is carried out, and the result of the test must be determined by the reading of the microplate reader.
8. All samples, washings and various wastes should be treated as infectious materials.
9. The different batch components of this reagent must not be mixed.
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