Rat erythropoietin (EPO) enzyme-linked immunoassay kit instruction manual Xiamen Huijia Biotechnology Co., Ltd. This kit is for research use only. Drug name: Generic name: Rat erythropoietin (EPO) enzyme-linked immunoassay kit Purpose of use: This kit is used to determine the content of erythropoietin (EPO) in rat serum, plasma, or other tissue fluids. Experimental principle This kit uses the double antibody sandwich method to determine the level of rat erythropoietin (EPO) in the specimen. The microplate was coated with purified anti-erythropoietin (EPO) antibody to make a solid-phase antibody, and rat erythropoietin (EPO) was added to the monoclonal antibody-coated microwells in turn, followed by HRP-labeled erythropoiesis EPO 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 the erythropoietin (EPO) in the sample. The absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm, and the concentration of rat erythropoietin (EPO) in the sample was calculated by a standard curve. Kit composition 1 20-fold concentrated washing solution 30ml × 1 bottle 7 Stop solution 6ml × 1 bottle 2 Enzyme label reagent 6ml × 1 bottle 8 Standard (27IU / 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. 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. Operating steps Dilution and sample loading of standards: set up 10 wells of the standard on the enzyme-coated plate, add 100 μl of the standard in the first and second wells, and then add the standard 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 amount of sample added to each well is 50 μl, and the concentrations are (18 IU / L, 12 IU / L, 6 IU / L, 3 IU / L, 1.5 IU / L). Sample addition: set blank wells ( Blank control wells do not add samples and enzyme labeling reagents, the rest of the operation is the same), the sample well to be tested. Add 40μl of sample diluent to the sample wells to be tested on the enzyme-coated plate, and then add 10μl of the sample to be tested (sample final The dilution 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, gently shake to mix. Incubation: seal the plate with the sealing plate and incubate at 37 ℃ for 30 minutes. Solution: Dilute the 20-fold concentrated washing solution with distilled water 20-fold and wash for later use: Carefully remove the sealing film, discard the liquid, spin dry, fill each well with the washing solution, let it sit for 30 seconds, then discard, repeat 5 times , Pat dry. Add enzyme: add 50μl of enzyme label reagent to each well, except the blank well. Incubate: operate the same as 3. Wash: operate the same as 5. Color development: add the developer A50μl first to each well, then add the developer B50μl , Shake gently to mix, and develop color in the dark at 37 ° C for 15 minutes. Termination: add 50μl of stop solution to each well to stop the reaction (the blue color turns to yellow at this time) ). Determination: The absorbance (OD value) of each well is measured in sequence with the blank air conditioner at zero, 450 nm wavelength. The measurement should be performed within 15 minutes after the addition of the stop solution. The calculation takes the standard concentration as the abscissa and the OD value as the ordinate , Draw a standard curve on the graph 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 calculate the linear regression equation of the standard curve using the concentration and OD value of the standard, and then the sample The OD value 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. Notes 1. The kit should be taken out of the refrigerated environment and should be equilibrated at room temperature for 15-30 minutes before use. If it is not used up after being unsealed, the slats should be stored in a sealed bag. 2. The concentrated washing liquid may crystallize out, which can be heated and dissolved in a water bath when diluted, and the results will not be affected during washing. 3. Each Use a sampler for stepwise sample addition, and correct its accuracy frequently to avoid test errors. It is best to control the time of one sample addition within 5 minutes. If there are many specimens, it is recommended to use a rifle to add samples. Please measure each time Do the standard curve at the same time, it is best to do double wells. 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 the sample diluent by a certain multiple (n times) After the measurement, please multiply the total dilution factor (× n × 5) at the end of the calculation. Perform the operation in strict accordance with the instructions, and the test results must be determined based on the reading of the microplate reader. 6. The substrate should be kept away from light. 7. Seal The membrane 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. Components of different batches of this reagent should not be mixed. 10. If there is an English instruction Different, subject to the English manual. Detection range: 0.156 ng / ml-10 ng / ml Sensitivity: 0.039 ng / ml Specifications: 96 servings / box storage conditions and expiration date 1. Kit storage :; 2-8 ℃. 2. Validity: 6 months
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