Human interferon (IFN-γ) enzyme-linked immunoassay (ELISA)
Kit instruction manual
This kit is for research use only.
Drug Name:
Generic name: Human IFN-γ ELISA kit
purpose of usage:
This kit is used to determine the content of interferon-γ (IFN-γ) in human serum, plasma, or other related tissue fluids.
Experimental principle
This kit uses the double antibody sandwich method to determine the level of human interferon-γ (IFN-γ) in the specimen. Microporous plates were coated with purified human interferon-gamma (IFN-γ) antibody to make solid-phase antibodies. Interferon-gamma (IFN-gamma) was added to the monoclonal antibody-coated microwells in turn, followed by HRP-labeled sheep The anti-human receptor binds to form an antibody-antigen-enzyme-labeled antibody complex. After thorough washing, the substrate TMB is added for color development. TMB is converted into blue under the catalysis of HRP enzyme, and into the final yellow under the action of acid. The color depth is positively correlated with interferon gamma (IFN-γ) in the sample. The absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm, and the content of human interferon-γ (IFN-γ) in the sample was calculated by a standard curve.
Kit composition
1
20 times concentrated washing liquid
50ml × 1 bottle
7
Stop solution
6ml × 1 bottle
2
Enzyme reagent
6ml × 1 bottle
8
Standard product (150μg / L)
0.5ml × 1 bottle
3
Enzyme coated plate
12 holes × 8
9
Standard dilution
1.5ml × 1 bottle
4
Sample diluent
6ml × 1 bottle
10
Instructions
1 serving
5
Developer A liquid
6ml × 1 bottle
11
Sealing film
2 sheets
6
Developer B liquid
6ml × 1 / bottle
12
sealed bag
1
Specimen processing and requirements
1. Serum, plasma, cerebrospinal fluid, and abdominal cavity fluid samples can be directly measured;
2. The samples containing NaN3 cannot be detected because NaN3 inhibits horseradish peroxidase (HRP) activity.
3. Experiment as soon as possible after collection. If the experiment cannot be performed immediately, the specimen can be stored at -20 ° C, but repeated freezing and thawing should be avoided.
Steps
1. Dilution and loading of standard products: set 10 standard wells on the enzyme-coated plate, add 100 μl of the standard products in the first and second wells, and then add the standard products in the first and second wells. 50μl of diluent, mix well; then take 50μl of each in the first and second wells and discard; then add 50μl of each to the third and fourth wells, and then add standard to the third and fourth wells respectively to dilute 50μl of the solution, after mixing, then add 50μl to the fifth and sixth wells respectively, and then add 50ul of the standard dilution solution to the fifth and sixth wells respectively, and mix; Add 50μl of each of the six wells to the seventh and eighth wells, and then add 50μl of the standard dilution solution to the seventh and eighth wells respectively. After mixing, take 50μl from the seventh and eighth wells and add to In the ninth and tenth wells, add 50 μl of the standard dilution solution to the ninth and tenth wells respectively. After mixing, take 50 μl each from the ninth and tenth wells and discard. (After dilution, the volume of each well is 50μl, and the concentration is 100μg / L, 50μg / L, 25μg / L, 12.5μg / L, 6.25μg / L).
2. Add samples: set up blank wells (the blank control wells do not add samples and enzyme reagents, the rest of the steps are the same) and the sample wells to be tested. Add 40μl of sample diluent to the test sample well of the enzyme-coated plate, and then add 10μl of the sample to be tested (the final dilution of the sample 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 a sealing plate and incubate at 37 ° C for 30 minutes.
4. Mixing solution: dilute 20 times concentrated washing liquid with distilled water 20 times and reserve
5. Washing: Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with the washing liquid, let it stand for 30 seconds and then discard, repeat this 5 times, 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, then add 50μl of developer B, mix gently, and develop at 37 ° C in the dark for 15 minutes.
10. Termination: Add 50μl of stop solution to each well to stop the reaction (at this time the blue will turn to yellow).
11. Determination: Measure the absorbance (OD value) of each well in sequence with the blank air conditioner at zero and 450 nm wavelength. The measurement should be carried out within 15 minutes after adding the stop solution.
Calculation
Taking the concentration of the standard as the abscissa (logarithmic coordinate) and the OD value as the ordinate (ordinary coordinate), draw a standard curve on the graph paper, and find out the corresponding concentration from the standard curve according to the OD value of the sample; then multiply by Dilution factor; or calculate the linear regression equation of the standard curve using the concentration and OD value of the standard, substitute the OD value of the sample into the equation, calculate the sample concentration, and multiply it by the dilution factor to obtain 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. If the enzyme label coated plate is unopened, the strip should be stored in a sealed bag.
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. It is best to control the sampling time within 5 minutes. If there are many specimens, 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 (the OD value of the sample is greater than 1.5 times the OD value of the standard pore), please dilute it with a certain multiple of the sample diluent (n times) and then determine it. When calculating, please multiply the total dilution factor (× n × 5).
5. The sealing film is limited to one-time use to avoid cross-contamination.
6. Please keep the substrate away from light.
7. The determination of the test results must be based on the reading of the microplate reader.
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:
5μg / L -150μg / ml
specification:
96 servings / box
Storage conditions and validity period
1. Kit storage :; 2-8 ℃.
2. Validity: 6 months
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