Human 25-hydroxyvitamin D (25-OH-D) enzyme-linked immunoassay kit instructions for use This kit is for research use only.
Drug Name:
Generic name: Human 25-hydroxyvitamin D (25-OH-D) enzyme-linked immunoassay kit Purpose of use:
This kit is used to determine the content of 25-hydroxyvitamin D (25-OH-D) in human serum, plasma, or other tissue fluids.
Experimental principle This kit uses the double antibody sandwich method to determine the level of human 25-hydroxyvitamin D (25-OH-D) in the specimen. Microporous plates were coated with purified anti-25-OH-D antibody to make solid-phase antibodies. Human 25-hydroxyvitamin D (25-OH-D) was added to the microwells coated with mAb in turn, and then labeled with HRP The goat anti-human antibody 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 25 hydroxyvitamin D (25-OH-D) in the sample.
The absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm, and the human 25-hydroxyvitamin in the sample was calculated by a standard curve
D (25-OH-D) concentration.
Kit composition
1 20 times concentrated washing solution 30ml × 1 bottle 7 stop solution 6ml × 1 bottle
2 Enzyme label reagent 6ml × 1 bottle 8 standard (90μg / L) 0.5ml × 1 bottle
3 Enzyme label coated plate 12 wells × 8 strips 9 standard dilutions 1.5ml × 1 bottle
4 Sample diluent 6ml × 1 bottle 10 instructions 1 copy
5 Developer A solution 6ml × 1 bottle 11 2 sealing film
6 Developer B solution 6ml × 1 / bottle 12 sealed bag 1 specimen requirement
1. Experiment as soon as possible after specimen collection. Serum and plasma can be directly detected
2. Organization: After extraction according to relevant literature, take the supernatant for testing
3. The samples containing NaN3 cannot be detected because NaN3 inhibits horseradish peroxidase (HRP) activity.
4. The specimen can be stored at -20 ℃, 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 add 100μl from the first well and the second well respectively to the third well and the fourth well, then add 50μl of the standard dilution solution to the third well
Mix well; then take 50μl each in the third and fourth wells and discard them, then add 50μl in the fifth and sixth wells respectively, and then add 50ul of standard dilution in the fifth and sixth wells Mix; after mixing, take 50μl from the fifth and sixth wells and add them to the seventh and eighth wells respectively. Then add 50μl of the standard dilution solution to the seventh and eighth wells respectively. 7. Take 50μl from the eighth and eighth wells respectively and add it to the ninth and tenth wells. Then add 50μl of the standard dilution solution to the ninth and tenth wells respectively. After mixing, take 50μl from the ninth and tenth wells and discard. (After dilution, the volume of each well is 50μl,
The concentrations are (60μg / L, 40μg / L, 20μg / L, 10μg / L, 5μg / 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 test sample (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, and evenly.
. Incubation: Seal the plate with a sealing film and incubate at 37 ° C for 30 minutes.
. Mixing solution: dilute 20 times concentrated washing liquid with distilled water 20 times and reserve
. Washing: Carefully remove the sealing film, discard the liquid, spin dry, fill each well with the washing solution, let stand for 30 seconds, then discard and repeat 5 times, pat dry.
. Add enzyme: add 50μl of enzyme label reagent to each well, except for blank wells.
. Incubation: The operation is the same as 3.
. Washing: The operation is the same as 5.
. Color development: add 50μl of developer A to each well, then add 50μl of developer B, mix gently, 37 ℃
15 minutes.
0. Stop: add 50μl of stop solution to each well to stop the reaction (at this time, the blue color turns to yellow).
1. 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 performed within 15 minutes.
Calculate the standard concentration as the abscissa, the OD value is the ordinate, draw a standard curve on the coordinate paper, the root
For the D value, find the corresponding concentration from the standard curve; multiply it by the dilution factor; or use the linear regression equation of the concentration of the standard and the OD value curve, substitute the OD value of the sample into the equation, calculate the sample concentration, and then multiply by The actual concentration of the sample.
Matters. The kit should be taken out of the refrigerated environment and equilibrated at room temperature for 15-30 minutes before use. The enzyme-coated plate should be used up and the strip should be stored in a sealed bag.
. Crystals may be precipitated in the concentrated washing liquid, which can be heated and dissolved in the water bath during dilution, and the results will not be affected during washing. The sampler should be used at each step of sample addition, and the accuracy should be regularly checked to avoid test errors. A sample is controlled within 5 minutes. If there are many specimens, it is recommended to use a volley gun to add samples.
. 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 sample is larger than the OD value of the first well of the standard well), please dilute it with the sample diluent by a certain multiple (n times) and then multiply it by the total dilution multiple (× n × 5).
. Strictly follow the instructions, the test results must be determined by the microplate reader. Please keep the substrate away from light.
. The sealing film is limited to one-time use to avoid cross-contamination.
. All samples, washing liquids and various wastes should be treated as infectious agents.
. The components of different batches of this reagent shall not be mixed.
0. If there is any difference with the English manual, the English manual shall prevail.
Measuring range:
3μg / L–80μg / L
grid:
96 servings / box storage conditions and expiration date. Kit storage :; 2-8 ℃.
. Validity: 6 months
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