Enzyme Linked Immunoassay Kit for Crab Estradiol (E2) Instructions for Use This kit is for research use only. Detection range: 48T2pmol / L -80 pmol / L Purpose: This kit is used to determine the content of estradiol (E2) in the serum, plasma and related liquid samples of crabs. Experimental principle The kit uses the double antibody sandwich method to determine the level of estradiol (E2) in specimens. The microplate was coated with purified Estradiol (E2) antibody to make a solid-phase antibody. Estradiol (E2) was added to the monoclonal antibody-coated microwells in turn, followed by HRP labeled estradiol (E2) ) The 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 estradiol (E2) in the sample. The absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm, and the concentration of Estradiol (E2) in the sample was calculated by a standard curve. Kit composition 1 20-fold concentrated washing solution 20ml × 1 bottle 7 Stop solution 3ml × 1 bottle 2 Enzyme reagent 3ml × 1 bottle 8 Standard (160 pmol / L) 0.5ml × 1 bottle 3 Enzyme label coated plate 12 wells × 4 strips 9 Standard diluent 1.5ml × 1 bottle 4 Sample diluent 3ml × 1 bottle 10 Instructions 1 copy 5 Developer A solution 3ml × 1 bottle 11 Sealing film 2 sheets 6 Developer B solution 3ml × 1 / Bottle 12 Sealed bag 1 specimen requirement 1. 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 repeated freezing and thawing should be avoided. The samples containing NaN3 cannot be detected because NaN3 inhibits horseradish peroxidase (HRP) activity. Procedure 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. 80 pmol / L No. 5 standard 150 μl original standard added 150 μl standard dilution 40 pmol / L No. 4 standard 150 μl No. 5 standard added 150 μl standard dilution 20 pmol / L No. 3 standard 150 μl Standard No. 4 added 150 μl standard dilution 10 pmol / L Standard No. 2 150 μl standard No. 3 added 150 μl standard dilution 5 pmol / L Standard No. 1 150 μl standard No. 2 added 150 μl standard dilution 2. Sample addition: set up blank wells (the blank control wells do not add samples and enzyme reagents, the rest of the operation is the same), standard wells, sample wells to be tested. Accurately add 50μl of the standard on the enzyme-coated plate, add 40μl of sample diluent to the sample well, 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 plate and incubate at 37 ° C for 30 minutes. 4. Mixing solution: dilute 20-fold concentrated washing solution with distilled water 20-fold and reserve for use 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, then add 50μl of developer B, mix gently, and develop color at 37 ° C in the dark 15 minutes. 10. Stop: Add 50μl of stop solution to each well to stop the reaction (the blue color turns to yellow at this time). 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. Summary of operating procedures: Calculate the concentration of the standard as the abscissa and the OD 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 by the dilution factor; or Calculate the linear regression equation of the standard curve using the concentration and OD value of the standard, 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. 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. 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 × 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 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. Storage conditions and validity period Kit storage :; 2-8 ℃. 2. Validity: 6 months
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