Detection range: 96T
1pg/ml-40pg/ml
purpose of usage:
This kit is used to determine the chemerin content in rat serum, plasma and related liquid samples.
Experimental principle
This kit uses the double antibody sandwich method to determine the level of rat chemerin in the specimen. The microplate was coated with purified rat chemerin antibody to prepare a solid phase antibody, and chemerin was sequentially added to the microcapsule of the coated monoclonal antibody, and then combined with the HRP-labeled chemerin antibody to form an antibody-antigen-enzyme-labeled antibody complex. After thorough washing, add the substrate TMB to develop color. TMB is converted to blue under the catalysis of HRP enzyme and converted to the final yellow color by the action of an acid. The color depth is positively correlated with chemerin in the sample. The absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm, and the concentration of rat chemerin in the sample was calculated from a standard curve.
Kit composition
1 | 30 times concentrated washing solution | 20ml × 1 bottle | 7 | Stop solution | 6ml × 1 bottle |
2 | Enzyme standard reagent | 6ml × 1 bottle | 8 | Standard product (80pg/ml) | 0.5ml × 1 bottle |
3 | Enzyme label coated plate | 12 holes × 8 | 9 | Standard dilution | 1.5ml × 1 bottle |
4 | Sample diluent | 6ml × 1 bottle | 10 | Instruction manual | 1 copy |
5 | Developer A solution | 6ml × 1 bottle | 11 | Sealing film | 2 sheets |
6 | Developer B solution | 6ml × 1 bottle | 12 | sealed bag | 1 |
Specimen requirements
1. The specimens should be extracted as soon as possible after collection, and the extraction should be carried out according to the 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.
2. Samples containing NaN3 could not be detected because NaN3 inhibited horseradish peroxidase (HRP) activity.
Steps
1. Dilution of standard: This kit provides one original standard, which can be diluted in a small tube according to the following chart.
40pg/ml | Standard No. 5 | Add 150 μl of standard dilution to 150 μl of the original standard |
20pg/ml | Standard No. 4 | Add 150 μl of standard dilution to 150 μl of standard #5 |
10pg/ml | Standard No. 3 | Add 150 μl of standard dilution to 150 μl of standard #4 |
5pg/ml | Standard 2 | Add 150 μl of standard dilution to 150 μl of Standard #3 |
2.5pg/ml | Standard No. 1 | 150 μl of Standard 2 is added to 150 μl of standard dilution |
2. Adding samples: set blank holes separately (the blank control wells are not added with the sample and the enzyme standard reagent, the other steps are the same), the standard holes, and the sample holes to be tested. Accurately load 50 μl of the standard on the enzyme-labeled plate, add 40 μl of the sample dilution to the well to be tested, and then add 10 μl of the sample to be tested (the final dilution of the sample is 5 times). Add the sample to the bottom of the well of the microplate, try not to touch the wall of the well, and shake gently to mix.
3. Incubation: The plate was sealed with a sealing film and incubated at 37 ° C for 30 minutes.
4. Liquor: 30 times concentrated washing solution diluted with distilled water 30 times and used
5. Washing: Carefully remove the sealing film, discard the liquid, dry it, fill each well with the washing solution, let stand for 30 seconds, then discard it, repeat 5 times, and pat dry.
6. Add enzyme: Add 50 μl of enzyme labeling reagent to each well, except for blank wells.
7. Incubation: The operation is the same as 3.
8. Wash: Operate the same as 5.
9. Color development: add 50 μl of color developer A, and then add 50 μl of color developer B, gently shake and mix, and color for 15 minutes at 37 °C.
10. Termination: 50 μl of stop solution was added to each well to stop the reaction (the blue color turned yellow).
11. Measurement: The absorbance (OD value) of each well was measured in sequence with a blank air conditioner of zero and a wavelength of 450 nm. The measurement should be carried out within 15 minutes after the addition of the stop solution.
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