Ningbo DOKEE Medical Technology Co., Ltd. , https://www.dokeemedical.com
Detection range: 96T
7μg/L -200μg/L
purpose of usage:
This kit is used to determine the Cortisol content in monkey serum, plasma and related liquid samples.
Experimental principle
The Cortisol kit was used to determine the level of cortisol in monkeys using a double antibody sandwich assay. The microplate was coated with purified monkey cortisol antibody to prepare a solid phase antibody, and cortisol was sequentially added to the microcapsules of the coated monoclonal antibody, and then combined with HRP-labeled cortisol antibody. The antibody-antigen-enzyme-labeled antibody complex is formed, and after thorough washing, the substrate TMB is added 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. The color depth is positively correlated with cortisol in the sample. Using a microplate reader
The absorbance (OD value) was measured at a wavelength of 450 nm, and the concentration of cortisol in the sample was calculated from the standard curve.
Kit composition
1 30 times concentrated washing solution 20ml × 1 bottle 7 stop solution 6ml × 1 bottle
2 enzyme standard reagent 6ml × 1 bottle 8 standard (400μg / L) 0.5ml × 1 bottle
3 enzyme label coating plate 12 holes × 8 strips 9 standard dilutions 1.5ml × 1 bottle
4 sample dilution 6ml × 1 bottle 10 instructions 1 copy
5 developer A liquid 6ml × 1 bottle 11 sealing film 2 sheets
6 developer B liquid 6ml × 1 bottle 12 sealed bag 1 specimen request
1. The specimens should be extracted as soon as possible after collection, and the extraction should be carried out according to the relevant literature. The experiment should be carried out as soon as possible after extraction. If the test cannot be performed immediately, the specimen can be stored at -20 °C, but repeated freezing and thawing should be avoided.
2. Samples containing NaN3 could not be detected because NaN3 inhibited horseradish peroxidase (HRP) activity.
Steps
1. Dilution of standard: This kit provides one original standard, which can be diluted in a small tube according to the following chart.
200μg/L No. 5 standard 150μl original standard added 150μl standard dilution
100 μg/L No. 4 Standard 150 μl of No. 5 Standard Add 150 μl Standard Diluent
50μg/L No. 3 Standard 150μl Standard No. 4 Add 150μl Standard Diluent
25μg/L No. 2 Standard 150μl No. 3 Standard Add 150μl Standard Diluent
12.5μg/L No. 1 Standard 150μl No. 2 Standard Add 150μl Standard Diluent
2. Adding samples: set blank holes separately (the blank control wells are not added with samples and enzyme labeling reagents, the other steps are the same), standard holes,
Sample hole to be tested. Accurately load 50 μl of the standard on the enzyme-labeled plate, and add 40 μl of the sample dilution to the sample well.
Then add 10 μl 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. Liquor: 20 times concentrated washing solution diluted with distilled water 20 times and used
5. Washing: Carefully remove the sealing film, discard the liquid, dry it, fill each well with the washing solution, let stand for 30 seconds, then discard it, repeat 5 times, and pat dry.
6. Add enzyme: Add 50 μl of enzyme labeling reagent to each well, except for blank wells.
7. Incubation: The operation is the same as 3.
8. Wash: Operate the same as 5.
9. Color development: add 50 μl of color developer A50 to each well, then add 50 μl of color developer B, gently shake and mix, and avoid coloration at 37 °C.
15 minutes.
10. Termination: 50 μl of stop solution was added to each well to stop the reaction (the blue color turned yellow).
11. Measurement: The absorbance (OD value) of each well was measured sequentially with a blank air conditioner of zero and a wavelength of 450 nm. The measurement should be carried out within 15 minutes after the addition of the stop solution.
Calculate the concentration of the standard as the abscissa and the OD as the ordinate. Draw a standard curve on the coordinate paper, according to the sample.
The OD value is determined from the standard curve by the corresponding concentration; multiplied by the dilution factor; or the linear regression equation of the standard curve is calculated from the concentration of the standard and the OD value, and the OD value of the sample is substituted into the equation to calculate the sample concentration, and then multiplied In dilution factor,
This is the actual concentration of the sample.
Precautions
1. The kit should be taken out from the refrigerated environment and allowed to equilibrate for 15-30 minutes at room temperature. If the enzyme label is unsealed after unsealing, the strip 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 well), please first dilute the sample dilution with a certain multiple (n times) and then measure it. When calculating, please multiply the total dilution by the total dilution. Multiple (×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 the instructions of the manual, the test results must be based on the microplate reader reading.
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.
10. In the case of an English manual, the English manual shall prevail.
Storage conditions and expiration date
1. The kit is stored at: 2-8 °C.
2. Validity: 6 months
Monkey Cortisol Kit Instructions
The Cortisol kit for monkeys is for research use only.