Manual of Mouse Prostaglandin E2 (PGE2) ELISA Kit
This kit is for research use only.
Drug Name:
Generic Name: Mouse Prostaglandin E2 (PGE2) ELISA Kit
purpose of usage:
This kit is used to determine the content of prostaglandin E2 (PGE2) in mouse serum, plasma and related liquid samples.
Experimental principle
This kit uses the enzyme-linked immunocompetitive method to determine the level of mouse prostaglandin E2 (PGE2) in the specimen. A microplate was coated with purified mouse prostaglandin E2 (PGE2) antibody to make a solid-phase antibody. Prostaglandin E2 (PGE2) and HRP-labeled goat anti-mouse antigen were added to the monoclonal antibody-coated microwells. They are competitively combined, and after thorough washing, substrate TMB is added for color development. The color of the sample is inversely related to the content of prostaglandin E2 (PGE2) in the sample. The absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm, and the content of mouse prostaglandin E2 (PGE2) in the sample was calculated by a standard curve.
Kit composition
1
20 times concentrated washing liquid
30ml × 1 bottle
7
Stop solution
6ml × 1 bottle
2
Enzyme reagent
6ml × 1 bottle
8
Standard product (450ng / 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 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.
Steps
1. Dilution and loading of standard products: set 10 standard wells on the enzyme-coated plate, add 100 μl of standard products in the first and second wells, and then add standard products in the first and second wells 50μl of diluent, mix well; then take 100μl from the first well and the second well and add them to the third and fourth wells respectively, and then add 50μl of standard diluent to the third and fourth wells respectively, mix well; Then take 50μl each in the third and fourth wells and discard it, then add 50μl each to the fifth and sixth wells, and then add 50ul of the standard dilution solution to the fifth and sixth wells respectively, and mix well; 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. Take 50μl from the eight wells and add them to the ninth and tenth wells. Then add 50μl of the standard dilution solution to the ninth and tenth wells. After mixing, take 50μl from the ninth and tenth wells and discard. (After dilution, the volume of each well is 50 μl, and the concentrations are 300 ng / L, 200 ng / L, 100 ng / L, 50 ng / L, 25 ng / L).
2. Adding 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 on 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, shake gently to mix.
3. Add enzyme: add 50μl of enzyme label reagent to each well, except for blank wells.
4. Incubation: seal the plate with a sealing film and incubate at 37 ° C for 60 minutes.
5. Mixing solution: dilute 20 times concentrated washing solution with distilled water 20 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. 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.
8. Termination: Add 50μl of stop solution to each well to terminate the reaction (at this time, the blue color turns to yellow).
9. Determination: The absorbance (OD value) of each well is measured in sequence with a blank air conditioner at zero, 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 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 it 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 substance to be tested in the specimen is too high (the OD value of the sample is less than the OD value of the first well of the standard well), please dilute it with a certain multiple (n times) of the sample diluent before measuring, and finally multiply the total dilution when calculating 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.
examination range:
20ng / L -400ng / L
specification:
96 servings / box
Storage conditions and validity period
1. Kit storage :; 2-8 ℃.
2. Validity: 6 months
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