Mouse leptin ELISA kit instructions
This kit is for research use only.
Drug Name:
Generic name: Mouse leptin ELISA kit
purpose of usage:
This kit is used to determine the content of leptin in mouse serum, plasma, or related tissue fluids.
Experimental principle
This kit uses the double antibody sandwich method to determine the level of leptin in mice. Microporous plates were coated with purified leptin antibody to make solid-phase antibodies. Leptin was added to the monoclonal antibody-coated microwells and combined with HRP-labeled goat anti-mouse antibody to form antibodies Antigen-enzyme labeled antibody complex, after thorough washing, add substrate TMB to develop color. 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 leptin in the sample. The absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm, and the content of mouse leptin 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 (1350ng / 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 and plasma samples can be directly measured;
2. Tissue: Take 1g of related tissues in 5ml PBS and homogenize. After incubating at room temperature for 5 hours (vortex mixing from time to time), 2000-3000 rpm / separation heart for 10 minutes, take the supernatant for testing.
3. The samples containing NaN3 cannot be detected because NaN3 inhibits horseradish peroxidase (HRP) activity.
4. 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: 10 standard wells are set in sequence on the enzyme-coated plate, add 100 μl of standard products in the first and second wells, and then add in the first and second wells Standard diluent 50μl, mix well; then add 100μl each to the third and fourth wells, and then add standard diluent 50μl to the third and fourth wells, after mixing, in the third well and the first Take 50μl each of the four wells and discard it; then add 50μl each to the fifth and sixth wells; add 50ul of the standard dilution solution to the fifth and sixth wells respectively and mix well; Take 50μl from the sixth well and add it to the seventh and eighth wells respectively; add 50μl of the standard dilution solution to the seventh and eighth wells respectively. After mixing, take 50μl from the seventh and eighth wells respectively. Go to the ninth and tenth wells; 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, and the concentrations are 900 ng / L, 600 ng / L, 300 ng / L, 150ng / L, 75 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. 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 5 times and 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 terminate the reaction (at this time, the blue color turns 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 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 test substance in the sample is too high (the OD value of the sample is greater than the OD value of the first well of the standard product), 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. Strictly follow the instructions, and the test results must be determined by the reading of the microplate reader.
6. The components of different batches of this reagent shall not be mixed.
7. The sealing film is limited to one-time use to avoid cross-contamination.
8. All samples, washing liquids and various wastes should be treated as infectious agents.
9. Please keep the substrate away from light.
examination range:
50ng / L -1000ng / L
specification:
96 servings / box
Storage conditions and validity period
1. Kit storage :; 2-8 ℃.
2. Validity: 6 months
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