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Manual of Cortisol ELISA Kit
This kit is for research use only.
Detection range: 96T
0pg / ml-160pg / ml
purpose of usage:
This kit is used to determine the content of Cortisol in human serum, plasma and related liquid samples.
Experimental principle
This kit uses the double antibody sandwich method to determine the level of human cortisol (Cortisol) in the specimen. The microtiter plate was coated with purified human cortisol antibody to make solid phase antibody. Cortisol (Cortisol) was added to the monoclonal antibody-coated microwells in turn, and then combined with HRP-labeled cortisol (Cortisol) antibody To form an antibody-antigen-enzyme-labeled antibody complex, and 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 Cortisol in the sample. The absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm, and the concentration of human cortisol (Cortisol) in the sample was calculated by a standard curve.
Kit composition
1
30 times concentrated washing solution
20ml × 1 bottle
7
Stop solution
6ml × 1 bottle
2
Enzyme reagent
6ml × 1 bottle
8
Standard product (160pg / ml)
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 requirements
1. Specimens are extracted as soon as possible after collection, and extraction is performed according to relevant literature, and experiments should be conducted as soon as possible after extraction. If the test cannot be performed immediately, the specimen can be stored at -20 ℃, but repeated freezing and thawing should be avoided
2. The samples containing NaN3 cannot be detected because NaN3 inhibits horseradish peroxidase (HRP) activity.
3. Serum:
After the blood coagulates naturally at room temperature for 10-20 minutes, centrifuge for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. If a precipitate forms during storage, it should be centrifuged again.
4. Plasma:
EDTA, sodium citrate or heparin should be selected as the anticoagulant according to the requirements of the specimen, mixed for 10-20 minutes, and centrifuged for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. If a precipitate forms during storage, it should be centrifuged again.
5. Urine:
Collect with sterile tubes. Centrifuge for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. If a precipitate forms during storage, it should be centrifuged again. Pleural and ascites, cerebrospinal fluid refer to this practice.
6. Cell culture supernatant:
When detecting secreted components, collect them with sterile tubes. Centrifuge for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully.
7. Culture cells
When detecting the components inside the cells, dilute the cell suspension with PBS (PH7.2-7.4), and the cell concentration will reach about 1 million / ml. By repeatedly freezing and thawing or adding tissue protein extraction reagents, the cells are destroyed and the intracellular components are released. Centrifuge for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. If a precipitate forms during storage, it should be centrifuged again.
8. Organize specimens
After cutting the specimen, weigh it. Add a certain amount of PBS, PH7.4. Quickly freeze and save with liquid nitrogen for later use. After the specimen melts, it still maintains a temperature of 2-8 ° C. Add a certain amount of PBS (PH7.4), or tissue protein extraction reagent, and homogenize the specimen by hand or homogenizer. Centrifuge for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. After aliquoting, a portion is to be tested, and the rest is frozen for future use.
Bring your own materials
1. Distilled water.
2. Sampler: 5ul, 10ul, 50ul, 100ul, 200, 500ul, 1000ul.
3. Oscillator and magnetic stirrer, etc.
Steps
1. Dilution of standard products: This kit provides one original standard product. The user can perform dilution in a small test tube according to the following chart.
2.
80pg / ml
Standard No. 5
150μl of original standard is added to 150μl standard dilution
40pg / ml
Standard 4
150μl Standard No. 5 is added to 150μl standard dilution
20pg / ml
Standard 3
150μl Standard No. 4 is added to 150μl Standard Diluent
10pg / ml
Standard No. 2
150μl Standard No. 3 is added to 150μl Standard Diluent
5pg / ml
Standard 1
150μl Standard No. 2 is added to 150μl Standard Diluent
3. Sample addition: set up blank wells (without adding samples and enzyme reagents in the blank control wells, the rest of the steps are the same), standard wells, and sample wells to be tested. Accurately add 50μl of the standard on the enzyme-coated plate, add 40μl of sample diluent to the sample well, 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.
4. Incubation: seal the plate with a sealing film and incubate at 37 ° C for 30 minutes.
5. Mixing solution: Dilute 30 times concentrated washing liquid with distilled water 30 times and reserve
6. Washing: Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with the washing solution, let it stand for 30 seconds and discard, repeat 5 times, pat dry.
7. Add enzyme: add 50μl of enzyme label reagent to each well, except for blank wells.
8. Incubation: The operation is the same as 3.
9. Washing: The operation is the same as 5.
10. Color development: add 50μl of developer A to each well, and then add 50μl of developer B, mix gently, and develop for 15 minutes in the dark at 37 ℃.
11. Stop: add 50μl of stop solution to each well to stop the reaction (at this time, the blue will turn to yellow).
12. Determination: The absorbance (OD value) of each well is measured sequentially with zero air conditioner and 450nm wavelength. The measurement should be carried out within 15 minutes after adding the stop solution.
Summary of operating procedures:
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, and find the corresponding concentration from the standard curve according to the OD value of the sample; then multiply by the dilution factor; Calculate the linear regression equation of the standard curve with the OD value, 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 the OD value of the first well of the standard well), please first dilute it with a certain multiple (n times) of the sample diluent and then determine it. When calculating, please multiply the total dilution Multiple (× 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. Strictly follow the instructions, and the test results must be determined by 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.
performance
1. Sensitivity: The smallest detection concentration is less than No. 1 standard. Linearity of dilution. The correlation coefficient R between the linear regression of the sample and the expected concentration is 0.990.
2. Specificity: Does not react with other human cytokines.
3. Repeatability: The coefficients of variation within and between plates are less than 10%.
4. Limitations: The results above the No. 6 standard product are non-linear, and accurate results cannot be obtained according to this standard curve.
Storage conditions and validity period
1. Kit storage :; 2-8 ℃.
2. Validity: 6 months