Human immunoglobulin A (IgA) enzyme-linked immunoassay (ELISA) Kit instruction manual This kit is for research use only. Generic name: Human immunoglobulin A (IgA) ELISA kit purpose of usage: This kit is used to determine the content of immunoglobulin A (IgA) in human serum, plasma, or other tissue fluids. Experimental principle: This kit uses the double antibody sandwich method to determine the level of human immunoglobulin A (IgA) in the specimen. The microplate was coated with purified anti-IgA antibody to make a solid phase antibody. Human immunoglobulin A (IgA) was added to the monoclonal antibody-coated microwells in turn, and then combined with HRP-labeled goat anti-human antibody to form The antibody-antigen-enzyme-labeled antibody complex is thoroughly washed and added with substrate TMB 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 human immunoglobulin A in the sample. The absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm, and the concentration of human immunoglobulin A (IgA) in the sample was calculated by a standard curve. Kit composition: 125 times concentrated washing solution 20ml × 1 bottle 7 stop solution 3ml × 1 bottle 2 Enzyme label reagent 3ml × 1 bottle 8 standard products (13.5μg / ml) 0.5ml × 1 bottle 3 Enzyme label coating plate 12 well × 4 strips 9 standard dilutions 1.5ml × 1 bottle 4 Sample diluent 10ml × 1 bottle 10 instructions 1 copy 5 developer A solution 3ml × 1 bottle 11 sealing film 2 sheets 6 Developer B liquid 3ml × 1 / bottle 12 sealed bags 1 Specimen requirements: 1. Experiment as soon as possible after specimen collection. 2. 1: 200 dilution of serum to be tested: specifically, dilute the serum to be tested by 1:20 in the test tube (190μl sample dilution) Add 10μl of serum to be tested to the solution and mix well), then dilute the serum to be tested by 1:10 (take 10μl of 1:20 diluted Add 90μl of sample diluent to the serum to be tested and mix well) 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. Steps: 1. Dilution and loading of standard products: 10 standard wells are set on the enzyme-coated plate, 100μl of the standard products are added to the first and second wells respectively, and then the standard products are added to 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 50μl, and the concentration is (9μg / ml, 6μg / ml, 3μg / ml, 1.5μg / ml, 0.75μg / ml). 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 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. 3. Incubation: seal the plate with a sealing film 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 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, and 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 stop 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. 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; or use the standard 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. 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 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 × 1000). 5. Strictly follow the instructions, and the test results must be determined by the reading of the microplate reader 6. Please keep the substrate away from light. 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. 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. Detection range: 0.5μg / ml-10μg / ml Specification: 96 servings / box Storage conditions and validity period: 1. Kit storage :; 2-8 ℃. 2. Validity: 6 months Shanghai Yueyan Biological Technology Co., Ltd. is one of the main suppliers of immunology products in China. It provides various original and sub-packaged detection ELISA kits with high quality and low price, and complete after-sales service. Any problem with the kit has technology The teacher will help you out. It can be tested on behalf of you for free, so as to serve you better. If you have any other questions, please contact us in time.
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Instruction manual of human immunoglobulin A (IgA) ELISA kit