Human JE antigen (JEV-Ag) Elisa kit

Human JE-Ag (JEV-Ag) Elisa Kit Instructions The human JE-Ag (Elisa) Kit is for research use only. Detection range: 96T 2.0 ng / L -48 ng / L Purpose: This kit is used to determine the content of JEV-Ag in human serum, plasma and related liquid samples. Experimental principle: Human JE-Ag (JEV-Ag) Elisa kit instructions This kit uses the double antibody sandwich method to determine the level of human JE-Ag (JEV-Ag) in the specimen. Coat the microplate with purified antibody to make a solid-phase antibody, add JEV-Ag (JEV-Ag) to the monoclonal antibody-coated microwells in turn, and then combine with HRP-labeled antibody to form an antibody-antigen-enzyme label 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 the JE-Ag in the sample. The absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm, and the concentration of human JE-Ag (JEV-Ag) in the sample was calculated by a standard curve. Human JE antigen (JEV-Ag) Elisa 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 (96ng / L) 0.5ml × 1 bottle 3. Enzyme label coated plate 12 wells × 8 strips 9 standard dilution 1.5ml × 1 bottle 4. sample dilution 6ml × 1 bottle 10 instructions 1 copy 5. developer A solution 6ml × 1 bottle 11 seals 2 sheets of film 6. Developer B solution 6ml × 1 / bottle 12 sealed bag 1 specimen requirements: 1. Cannot detect samples containing NaN3, because NaN3 inhibits horseradish peroxidase (HRP) activity. 2. The specimens should be extracted as soon as possible after collection. The extraction should be carried out according to relevant literature. The experiment should be carried out 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 procedures should be avoided: 1. Dilution of the standard: This kit provides one original standard, the user can follow the chart below for small Dilute in the test tube. 48 ng / L No. 5 standard 150μl original standard added 150μl standard diluent 24 ng / L No. 4 standard 150μl No. 5 standard added 150μl standard diluent 12 ng / L No. 3 standard 150μl Standard 4 added 150μl standard dilution 6.0 ng / L Standard 2 150μl standard 3 added 150μl standard dilution 3.0 ng / L Standard 1 150μl standard 2 added 150μl standard dilution 2. Add sample: 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 30 times concentrated washing solution with distilled water 30 times before use. 5. Washing: Carefully remove the sealing film, discard the liquid, spin dry, fill each well with washing solution, and let it sit for 30 seconds. , Repeat this 5 times, pat dry. 6. Add enzyme: Add 50μl of enzyme label reagent to each well, except for blank wells. 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 ℃ 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 zero air conditioner and 450nm wavelength. The measurement should be carried out within 15 minutes after adding the stop solution. Summary of operating procedures: calculation, with 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; 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 then multiply it by the dilution factor to obtain the actual concentration of the sample. Note 1. The kit should be taken out of the refrigerated environment and equilibrated at room temperature for 15-30 minutes before use. If the enzyme label coated plate is unopened 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 substance to be tested 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 the total dilution when calculating 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. Storage conditions and expiration date: 1. Kit storage: 2-8 ℃. 2. Validity: 6 months

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