Instruction Manual of Human Agrin ELISA Kit
This kit is for research use only.
Generic name: Human Agrin ELISA Kit
purpose of usage:
This kit is used to determine the content of Agrin in human serum, plasma, or other related tissue fluids.
This kit uses the double antibody sandwich method to determine the level of human Agrin in the specimen. The microplate is coated with purified Agrin antibody to make a solid phase antibody. Agrin antigen 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 Agrin in the sample. The absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm, and the concentration of human agrin (Agrin) in the sample was calculated by a standard curve.
20 times concentrated washing liquid
50ml Ã— 1 bottle
6ml Ã— 1 bottle
6ml Ã— 1 bottle
Standard product (225Î¼g / L)
0.5ml Ã— 1 bottle
Enzyme coated plate
12 holes Ã— 8
1.5ml Ã— 1 bottle
6ml Ã— 1 bottle
Developer A liquid
6ml Ã— 1 bottle
Developer B liquid
6ml Ã— 1 / bottle
Specimen processing and requirements
1. Serum, plasma, cerebrospinal fluid, and abdominal cavity fluid samples can be directly measured;
2. The samples containing NaN3 cannot be detected because NaN3 inhibits horseradish peroxidase (HRP) activity.
3. Experiment as soon as possible after collection. If the experiment cannot be performed immediately, the specimen can be stored at -20 Â° C, but repeated freezing and thawing should be avoided.
1. Dilution and loading of standard products: set 10 standard wells on the enzyme-coated plate, add 100 Î¼l of the standard products in the first and second wells, and then add the standard products in the first and second wells. 50Î¼l of diluent, mix well; then add 100Î¼l to the third and fourth wells respectively, and then add 50Î¼l of standard diluent to the third and fourth wells respectively, after mixing, first in the third and fourth wells Take 50Î¼l each and discard, then add 50Î¼l to the fifth and sixth wells respectively, and then add 50ul of standard dilution solution to the fifth and sixth wells respectively, mix well; Take 50Î¼l from each well and add it to the seventh and eighth wells, then add 50Î¼l of the standard dilution solution to the seventh and eighth wells respectively. After mixing, take 50Î¼l from the seventh and eighth wells respectively In the tenth and tenth wells, add 50 Î¼l of the standard dilution solution to the ninth and tenth wells respectively, and after mixing, take 50 Î¼l each from the ninth and tenth wells and discard. (After dilution, the amount of sample added to each well is 50 Î¼l, and the concentrations are 150 Î¼g / L, 100 Î¼g / L, 50 Î¼g / L, 25 Î¼g / L, and 12.5 Î¼g / L respectively).
2. Add 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 40Î¼l of sample diluent to the test sample well of the enzyme-coated plate, and then add 10Î¼l 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 Â° 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 liquid, let it stand for 30 seconds and then discard, repeat this 5 times, pat dry.
6. Add enzyme: add 50Î¼l 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 50Î¼l of developer A to each well, then add 50Î¼l of developer B, mix gently, and develop at 37 Â° C in the dark for 15 minutes.
10. Termination: Add 50Î¼l of stop solution to each well to stop the reaction (at this time the blue will turn 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.
Taking the concentration of the standard as the abscissa (logarithmic coordinate) and the OD value as the ordinate (ordinary coordinate), draw a standard curve on the graph paper, and find out the corresponding concentration from the standard curve according to the OD value of the sample; then multiply by 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.
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 (Ã— n Ã— 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 by 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.
10Î¼g / L -200Î¼g / L
96 servings / box
Storage conditions and validity period
1. Kit storage :; 2-8 â„ƒ.
2. Validity: 6 months
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