Rat Prostaglandin E2 (PGE2) Enzyme Linked Immunoassay (ELISA) Kit User Manual Xiamen Huijia Biotechnology Co., Ltd. This kit is for research use only. Drug name: Generic name: Rat prostaglandin E2 (PGE2) enzyme-linked immunoassay kit Purpose of use: This kit is used to determine the content of prostaglandin E2 (PGE2) in rat serum, plasma and related tissue samples. Experimental principle The kit uses the double antibody sandwich method to determine the level of rat prostaglandin E2 (PGE2) in the specimen. Microporous plates were coated with purified rat prostaglandin E2 (PGE2) antibody to make solid-phase antibodies. Prostaglandin E2 (PGE2) was added to the monoclonal antibody-coated microwells in turn, followed by HRP-labeled goat anti-mouse 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 color depth is positively correlated with prostaglandin E2 in the sample. The absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm, and the concentration of rat prostaglandin E2 (PGE2) in the sample was calculated by a standard curve. 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 (540ng / L) 0.5ml × 1 bottle 3 Enzyme label coated plate 12 well × 8 strips 9 standard diluent 1.5ml × 1 bottle 4 sample diluent 6ml × 1 bottle 10 instruction manual 1 copy 5 developer A solution 6ml × 1 bottle 11 sealing film 2 sheets 6 developer B solution 6ml × 1 / Bottle 12 sealed bag 1 specimen requirement 1. The samples containing NaN3 cannot be detected because NaN3 inhibits horseradish peroxidase (HRP) activity. 2. 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 the dilution and loading of the standard should be avoided in the repeated freeze-thaw operation steps: 10 standard wells are set on the enzyme label coating plate. Add 100 μl of the standard product to the second well, then add 50 μl of the standard dilution solution to the first and second wells, mix well; then take 100 μl from the first and second wells and add them to the third and third wells respectively Four wells, then add 50μl of standard dilution solution to the third and fourth wells respectively, and mix well; then take 50μl each of the third and fourth wells to discard, then add 50μl to the fifth and sixth wells respectively In the middle, add 50ul of the standard dilution solution to the fifth and sixth wells respectively, and mix; after mixing, take 50μl from the fifth and sixth wells respectively and add them to the seventh and eighth wells, and then add Add 50μl of standard dilution solution to the eighth and eighth wells respectively. After mixing, take 50μl from the seventh and eighth wells and add them to the ninth and tenth wells respectively. 50μl of the solution, 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 360ng / L, 240ng / L, 120ng / L, 60ng / L, 30ng / L). Sample addition: 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 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. Incubation: Seal the plate with a sealing film and incubate at 37 ° C for 30 minutes. Mixing solution: dilute 20 times concentrated washing solution with distilled water 20 times and then wash for standby: carefully peel off the sealing film, discard the liquid, spin dry, fill each well with washing solution, let stand for 30 seconds and then discard, repeat 5 Time, pat dry. Enzyme addition: 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, and develop at 37 ° C in the dark for 15 minutes. Termination: add 50μl of stopper solution to each well to stop the reaction (blue at this time) Li to yellow). Measurement: The absorbance (OD value) of each well was measured 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. Calculate the standard concentration 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; multiply by the dilution factor; The linear regression equation of the standard curve is calculated from the concentration and the OD value, the OD value of the sample is substituted into the equation, the sample concentration is calculated, and then multiplied by the dilution factor, which is the actual concentration of the sample. Matters needing attention 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. 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 first dilute it with a certain multiple (n times) of the sample diluent and then determine it. When calculating, please multiply the total dilution Multiple (× n × 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 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 it is different from the English manual, the English manual shall prevail. Detection range: 0.5 pg / ml-100 pg / ml Sensitivity: 0.25 pg / ml Two published foreign literature specifications: 96 copies / box storage conditions and expiration date 1. Kit storage :; 2-8 ℃. 2. Validity: 6 months
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