Human corticotropin-releasing hormone (CRH) ELISA kit instructions This reagent is for research use only: This kit is used to determine the content of human corticotropin-releasing hormone (CRH) in human serum, plasma and related liquid samples . Experimental principle: This kit uses the double antibody sandwich method to determine the level of human corticotropin-releasing hormone (CRH) in the specimen. The microplate was coated with purified human corticotropin-releasing hormone (CRH) antibody to make a solid-phase antibody, and the corticotropin-releasing hormone (CRH) was added to the monoclonal antibody-coated microwells in turn, followed by HRP-labeled corticotropin-releasing hormone (CRH) 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 corticotropin-releasing hormone (CRH) in the sample. The absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm, and the concentration of human corticotropin-releasing hormone (CRH) in the sample was calculated by a standard curve. Kit composition: kit composition 48 well configuration 96 well configuration storage instructions 1 copy 1 copy sealing film 2 pieces (48) 2 pieces (96) 1 sealed bag 1 enzyme coated plate 1 × 48 1 × 96 2 Store standard at -8 ° C: 1800ng / L 0.5ml × 1 bottle 0.5ml × 1 bottle at 2-8 ° C Store standard dilution 1.5ml × 1 bottle 1.5ml × 1 bottle at 2-8 ° C Store enzyme label reagent 3 ml × 1 bottle of 6 ml × 1 bottle of sample diluent at 2-8 ° C 3 ml × 1 bottle of 6 ml × 1 bottle of 2-8 ° C Developer A 3 ml × 1 bottle of 6 ml × 1 bottle of 2-8 ° C Developer B solution 3 ml × 1 bottle 6 ml × 1 bottle at 2-8 ° C storage stop solution 3ml × 1 bottle 6ml × 1 bottle at 2-8 ° C Concentrated washing solution (20ml × 20 times) × 1 bottle (20ml × 30 times) × 1 bottle at 2-8 ° C to store the sample. Processing and requirements: 1. Serum: room temperature blood is naturally coagulated for 10-20 minutes, centrifuged for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully and centrifuge again if a precipitate appears during storage. 2. Plasma: EDTA or sodium citrate should be selected as the anticoagulant according to the requirements of the specimen, mixed for 10-20 minutes, and centrifuged for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. If a precipitate forms during storage, it should be centrifuged again. 3. Urine: collected in a sterile tube and centrifuged for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. If a precipitate forms during storage, centrifuge again. Pleural and ascites, cerebrospinal fluid reference implementation. 4. Cell culture supernatant: When detecting secreted components, collect with a sterile tube. Centrifuge for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. When detecting the components inside the cells, dilute the cell suspension with PBS (PH7.2-7.4), and the cell concentration will reach about 1 million / ml. Through repeated freezing and thawing, the cells are destroyed and the intracellular components are released. Centrifuge for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. If a precipitate forms during storage, it should be centrifuged again. 5. Organize the specimen: after cutting the specimen, weigh it. Add a certain amount of PBS, PH7.4. Quickly freeze and save with liquid nitrogen for later use. After the specimen melts, it still maintains a temperature of 2-8 ° C. Add a certain amount of PBS (PH7.4) and homogenize the specimen with a manual or homogenizer. Centrifuge for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. After aliquoting, a portion is to be tested, and the rest is frozen for future use. 6. The specimen 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 should be avoided. 7. Samples containing NaN3 cannot be detected because NaN3 inhibits the activity of horseradish peroxidase (HRP). Operation steps: standard dilution and sample addition: set up 10 standard wells 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 the product dilution, mix well; then take 100μl from the first well and the second well respectively to the third and fourth wells, and then add 50μl of the standard dilution solution to the third and fourth wells respectively, mix well ; Then in the third and fourth wells, first take 50μl to discard, and then take 50μl respectively 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 eighth well and add it to the ninth and tenth wells. Then add 50μl of the standard dilution solution to the ninth and tenth wells respectively. After mixing, take 50μl from the ninth and tenth wells and discard. (After dilution, the amount of sample added to each well is 50μl, and the concentration is 1200ng / L, 800ng / L, 400ng / L, 200ng / L, 100ng / L. Sample addition: blank wells are set separately (the blank control wells do not add sample and Enzyme label reagent, the rest of the steps are the same), the sample well to be tested. First add 40μl of sample diluent to the sample well of the enzyme 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 the sealing plate and incubate at 37 ℃ for 30 minutes. Dosing: add 30 (48T) 20 times) concentrated washing solution diluted with distilled water 30 times (20 times of 48T) and use it after dilution. Washing: 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 Go, repeat this 5 times, pat dry. Add enzyme: add 50μl of enzyme label reagent to each well, except blank well. Incubate: operate the same as 3. Wash: operate the same as 5. Color development: add the developer A50μl first to each well, 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. Reaction (blue to yellow at this time). Determination: The absorbance (OD value) of each well is measured in sequence with a blank air conditioner at zero, 450 nm wavelength. The measurement should be performed within 15 minutes after the addition of the stop solution. Note: The kit is Take it out in a refrigerated environment and let it equilibrate at room temperature for 15-30 minutes before use. If the enzyme-labeled coated plate is not used up after opening, the slat should be stored in a sealed bag. The concentrated washing liquid may crystallize out, which can be diluted Heating and solubilizing in a water bath does not affect the results during washing. The sampler should be used at each step of sample addition, and its accuracy should be regularly checked to avoid test errors. The time of one sample addition is best controlled within 5 minutes, such as There are a large number of specimens, it is recommended to use a rifle to add samples. Please make a standard curve at the same time for each measurement, preferably 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 hole of the standard product) , Please dilute with a certain multiple (n times) of the sample diluent before measuring, and multiply the total dilution factor (× n × 5) in the calculation. The sealing film is only for one-time use to avoid cross contamination. Substrate Please protect from light. Follow strictly The operation of the instructions is carried out, and the determination of the test results must be based on the reading of the microplate reader. All samples, washing solutions and various wastes should be treated as infectious agents. The components of different batches of this reagent should not be mixed. 10. If there is an English instruction The difference is based on the English manual. Calculation: Take the concentration of the standard as the abscissa and the OD 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 Take the dilution factor; 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 multiply by the dilution factor to get the actual concentration of the sample. (This The figure is for reference only) Kit performance: 1. The linear regression coefficient of the sample and the expected concentration correlation coefficient R value is above 0.92. 2. Within and between batches should be less than 9% and 15% respectively. Storage conditions and expiration date: 1. Kit storage :; 2-8 ℃. 2. Validity: 6 months
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