Human P substance (SP) ELISA kit instructions This kit is for research use only.
Drug Name:
Generic name: Human P substance (SP) diagnostic kit Purpose of use:
This kit is used to determine the content of substance P (SP) in human serum, plasma, or other tissue fluids. Experimental principle This kit uses the double antibody sandwich method to determine the level of human substance P (SP) in the specimen. A microplate is coated with purified human substance P (SP) antibody to make a solid phase antibody. Substance P (SP) is added to the monoclonal antibody-coated microwells in turn, and then combined with HRP-labeled goat anti-human antibody 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 the substance P (SP) in the sample. The absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm, and the concentration of human substance P (SP) in the sample was calculated by a standard curve.
Kit composition 1
20 times concentrated washing liquid 50ml × 1 bottle 7
Stop solution 6ml × 1 bottle
2
Enzyme reagent 6ml × 1 bottle 8
Standard product (90ng / L)
0.5ml × 1 bottle
3
Enzyme label coating plate 12 well × 8 strips 9
Standard diluent 2ml × 1 bottle
4
Sample diluent 6ml × 1 bottle 10
Instructions 1
5
Developer A liquid 6ml × 1 bottle 11
Sealing film 2 sheets
6
Developer B liquid 6ml × 1 / bottle 12
1 sealed bag
Specimen requirements 1. Experiment as soon as possible after specimen collection. 2. The samples containing NaN3 cannot be detected because NaN3 inhibits horseradish peroxidase (HRP) activity. 3. The specimen can be stored at -20 ℃, but repeated freezing and thawing should be avoided. Steps
1. Dilution and loading of standard products: set 10 standard wells on the enzyme-coated plate, add 100 μl of standard products in the first and second wells, and then add standard products 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 concentration is (600 ng / L, 400 ng / L, 200 ng / L, 100 ng / L, 50 ng / L). 2. Sample addition: set blank respectively Wells (the blank control wells do not add samples and enzyme reagents, the rest of the operation is the same), the sample wells to be tested. First add 40μl of the sample diluent to the test wells of the enzyme coated plate, and then add 10μl of the sample The final dilution of the sample is 5 times). Add the sample and add the sample to the bottom of the well of the microtiter plate, 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 ℃ 30 minutes 4. Mixing solution: dilute 20 times concentrated washing solution with distilled water 20 times and reserve for use 5. Washing: carefully peel off the sealing film, discard the liquid, spin dry, fill each well with washing solution, and let stand for 30 seconds After discarding, repeat this 5 times and pat dry. 6. Add enzyme: add 50μl of enzyme labeling reagent to each well, except for the 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 for 15 minutes in the dark at 37 ° C. 10 Termination: add 50μl of stopper 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 450nm wavelength. Within 15 minutes after the 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 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. Strictly follow the instructions, and the test results must be determined based on 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. examination range:
40 ng / L -800 ng / L
specification:
96 servings / box storage conditions and expiration date
1. Kit storage :; 2-8 ℃. 2. Validity: 6 months Beijing Qisong Biological Technology Co., Ltd. is a professional agent for many well-known international life science brands. Committed to the sales and promotion of various ELISA kits, immunohistochemical kits, primary and secondary antibodies, cytokines, biological reagents, pipettes, and consumables. Company website: Order phone
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