Manual of human tumor necrosis factor alpha (TNF-α) ELISA kit
This kit is for research use only.
Drug Name:
Generic name: Human Tumor Necrosis Factor Alpha (TNF-α) ELISA Kit
purpose of usage:
This kit is used to determine the content of tumor necrosis factor alpha (TNF-α) in human serum, plasma, or other tissue fluids.
Experimental principle The kit uses the double antibody sandwich method to determine the level of human tumor necrosis factor α (TNF-α) in the specimen. With purified people
Tumor necrosis factor alpha (TNF-α) antibody is coated on the microplate to make a solid phase antibody, which is added to the microwells coated with monoclonal antibody in sequence
Tumor necrosis factor alpha, then combined with HRP-labeled goat anti-human antibody to form an antibody-antigen-enzyme-labeled antibody complex, after
After thorough washing, add substrate TMB to develop color. TMB turns into blue under the catalysis of HRP enzyme, and under the action of acid
Into the final yellow. The color depth is positively correlated with human tumor necrosis factor α in the sample. Use a microplate reader at 450nm
The absorbance (OD value) was measured under long time, and the concentration of human tumor necrosis factor α (TNF-α) in the sample was calculated by the standard curve.
Kit composition
1 20 times concentrated washing solution 50ml × 1 bottle 7 stop solution 6ml × 1 bottle
2 Enzyme label reagent 6ml × 1 bottle 8 standard (450ng / L) 0.5ml × 1 bottle
3 Enzyme-labeled coating plate 12 wells × 8 strips 9 Standard diluent 2ml × 1 bottle
4 Sample diluent 6ml × 1 bottle 10 Instructions 1 copy
5 Developer A solution 6ml × 1 bottle 11 sealing film 2 sheets
6 Developer B solution 6ml × 1 / bottle 12 sealed bag 1
Specimen requirements Experiment as soon as possible after specimen collection.
2. The samples containing NaN3 cannot be detected because NaN3 inhibits horseradish peroxidase (HRP) activity.
3. The specimen can be stored at -20 ℃, but repeated freezing and thawing should be avoided.
Steps
1. Dilution and sample addition of standard products: 10 standard wells are provided on the enzyme-coated plate, and the standard is added in the first and second wells respectively
100μl of quasi-standard, then add 50μl of standard diluent to the first and second wells, mix well; then from the first well and second
Take 100μl of each well and add them to the third and fourth wells respectively, then add 50μl of standard dilution solution to the third and fourth wells,
Mix well; then take 50μl each in the third and fourth wells and discard, then add 50μl each to the fifth and sixth wells respectively
In the middle, add 50ul of the standard dilution solution to the fifth and sixth wells respectively, mix well;
Add 50μl to the seventh and eighth wells respectively, then add 50μl of the standard dilution solution to the seventh and eighth wells respectively, mix
After smoothing, take 50μl from the seventh and eighth holes respectively and add them to the ninth and tenth holes, then add the standards to the ninth and tenth holes respectively
50μl of the product dilution, after mixing, take 50μl from the ninth and tenth wells and discard. (After dilution, the volume of each well is 50μl,
The concentrations are (300 ng / L, 200ng / L, 100ng / L, 50 ng / L, 25 ng / L).
2. Adding samples: set up blank wells separately (the blank control wells do not add samples and enzyme reagents, the rest of the steps are the same), the sample to be tested
Pinhole. Add 40μl of sample diluent to the test sample well on the enzyme-coated plate, and then add 10μl of the test sample (sample
The final dilution of the product is 5 times). Add the sample and add the sample to the bottom of the well of the microplate, try not to touch the wall of the well, shake gently to mix 3. Incubation: seal the plate with the sealing film and incubate for 30 minutes at 37 ℃
4. Mixing solution: dilute 20 times concentrated washing solution with distilled water 20 times and reserve
5. Washing: Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with washing liquid, let stand for 30 seconds and then discard, so
Repeat 5 times and pat dry.
6. Add enzyme: add 50μl of enzyme label reagent to each well, except blank well.
7. Incubation: The operation is the same as 3.
8. Washing: The operation is the same as 5.
9. Color development: add 50μl of developer A to each well, and then add 50μl of developer B, mix gently, and develop color at 37 ℃ in the dark
15 minutes.
10. Termination: Add 50μl of stop solution to each well to terminate the reaction (at this time, the blue color turns to yellow).
11. Determination: Measure the absorbance (OD value) of each well in sequence with the blank air conditioner at zero and 450nm wavelength. Determination should be terminated
Within 15 minutes after the solution.
Calculation
Taking the concentration of the standard as the abscissa and the OD value as the ordinate, draw a standard curve on the coordinate paper, according to the sample
The corresponding concentration of OD value is found by the standard curve; then multiplied by the dilution factor; or the standard concentration and OD value are used to calculate the standard
The linear regression equation of the quasi-curve, the OD value of the sample is substituted into the equation, the sample concentration is calculated, and then multiplied by the dilution factor,
This is the actual concentration of the sample.
Precautions
1. The kit should be equilibrated at room temperature for 15-30 minutes before being taken out of the refrigerated environment.
After use, the slats should be stored in sealed bags.
2. Crystals may be precipitated in the concentrated washing liquid, which can be heated and dissolved in a water bath during dilution, and the results will not be affected during washing.
3. The sampler should be used at each step of sample addition, and the accuracy should be regularly checked to avoid test errors. One sample loading time is best
Control within 5 minutes, if the number of specimens is large, it is recommended to use a volley gun to add samples.
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 test substance in the specimen is too high (sample OD value
Is greater than the OD value of the first well of the standard product), please dilute it with a certain multiple (n times) of the sample diluent before measuring.
When calculating, please multiply the total dilution factor (× n × 5).
5. Strictly follow the instructions, and the test results must be determined by the reading of the microplate reader
6. Please keep the substrate away from light.
7. The sealing film is limited to one-time use to avoid cross-contamination.
8. All samples, washing liquids and various wastes should be treated as infectious agents.
9. The components of different batches of this reagent shall not be mixed.
10. If there is any difference with the English manual, the English manual shall prevail.
examination range:
20ng / L -400 ng / L
specification:
96 servings / box
Storage conditions and validity period
1. Kit storage :; 2-8 ℃.
2. Validity: 6 months
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