This kit is for research use only. Detection range: 96T 0μg / L -8μg / L Purpose: This kit is used to determine the content of leptin (LEP) in rat serum, plasma and related liquid samples. Experimental principle: Rat leptin (LEP) Elisa kit instructions This kit uses the double antibody sandwich method to determine the level of rat leptin (LEP) in the specimen. Microporous plates were coated with purified rat leptin (LEP) antibody to make solid-phase antibodies, and leptin (LEP) was added to the monoclonal antibody-coated microwells in turn, followed by HRP labeled leptin (LEP) antibody The combination forms 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 shade of color is positively correlated with the leptin (LEP) in the sample. The absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm, and the concentration of rat leptin (LEP) 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 label reagent 6ml × 1 bottle 8 standard product (16μg / L) 0.5ml × 1 bottle; 3. enzyme label coating Plate 12 wells × 8 strips 9 standard dilutions 1.5ml × 1 bottle; 4. sample dilutions 6ml × 1 bottle 10 instructions 1 copy; 5. developer A solution 6ml × 1 bottle 11 sealing plate 2 sheets; 6 .Reagent B liquid 6ml × 1 / bottle 12 sealed bags 1; Specimen requirements: 1. Specimens should be extracted as soon as possible after collection. The extraction should be carried out according to relevant literature. Experiments should be conducted 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. The sample containing NaN3 cannot be detected because NaN3 inhibits the activity of horseradish peroxidase (HRP). Operation steps: 1. Dilution of standard product: This kit provides one original standard product. The user can perform dilution in a small test tube according to the following chart. 8μg / L No. 5 standard 150μl original standard added 150μl standard dilution; 4μg / L No. 4 standard 150μl No. 5 standard added 150μl standard dilution; 2μg / L No. 3 standard 150μl 4 Add 150μl standard diluent to standard # 1; 150μl standard 2 to 1μg / L # 150 add 150μl standard diluent; 0.5μg / L standard 1 to 150μl standard # 2 add 150μl standard diluent 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), standard wells, sample wells to be tested. Accurately add 50μl of standard on the enzyme-coated plate, add 40μl of sample diluent to the sample well, and then add 10μl of 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. 3. Incubation: seal the plate with a sealing film and incubate at 37 ° C for 30 minutes. 4. Mixing solution: Dilute the 30-fold concentrated washing solution with distilled water 30-fold and reserve for use. 5. Washing: Carefully remove the sealing film, discard the liquid, spin dry, fill each well with the washing solution, let it stand for 30 seconds and then discard , Repeat this 5 times, 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 color at 37 ° C in the dark 15 minutes. 10. Termination: Add 50 μl of stop solution to each well to stop the reaction (in this case, 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. Operation procedure summary: calculation: take 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; Or use the standard concentration and OD value to calculate the linear regression equation of the standard curve, 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. Note: 1. The kit should be equilibrated at room temperature for 15-30 minutes before it is taken out of the refrigerated environment. If the enzyme-coated plate is not used up after opening, 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 the water bath during dilution, and the results will not be affected during washing. 3. The sample adder should be used in each step of sample addition, and its 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 dilute it with a certain multiple of the sample diluent (n times) before measuring, and finally multiply by 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 based on the reading of the microplate reader. 8. All samples, washing liquid 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. Storage conditions and expiration date: 1. Kit storage: 2-8 ℃. 2. Validity: 6 months
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