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Product name: Rat C-peptide (CP) enzyme-linked immunoassay (ELISA) kit instruction manual
Specifications: original / 96T 48T / 96T
This kit is used to determine the content of C-peptide (CP) in rat serum, plasma and related liquid samples.
Specimen processing and requirements
1. Serum and plasma samples can be directly measured;
2. Urine, cerebrospinal fluid, and intraperitoneal fluid: After 2000-3000 rpm / separation of the heart for 10 minutes, take the supernatant for testing.
3. The samples containing NaN3 cannot be detected because NaN3 inhibits horseradish peroxidase (HRP) activity.
4. Experiment as soon as possible after collection. If the experiment cannot be performed immediately, the specimen can be stored at -20 ° C, but repeated freezing and thawing should be avoided.
Kit composition
1
20 times concentrated washing liquid
30ml × 1 bottle
7
Stop solution
6ml × 1 bottle
2
Enzyme reagent
6ml × 1 bottle
8
Standard product (270ng / L)
0.5ml × 1 bottle
3
Enzyme coated plate
12 holes × 8
9
Standard dilution
1.5ml × 1 bottle
4
Sample diluent
6ml × 1 bottle
10
Instructions
1 serving
5
Developer A liquid
6ml × 1 bottle
11
Sealing film
2 sheets
6
Developer B liquid
6ml × 1 / bottle
12
sealed bag
1
Bring your own items
1. 37 ℃ incubator
2. Standard specification microplate reader
3. Precision pipette and disposable tip
4. Distilled water
5. Disposable test tube
6. Absorbent paper
Steps
1. Dilution and sample addition of standard products: 10 standard wells are provided on the enzyme-coated plate, and the standard is added in the first and second wells respectively
100μl of quasi-standard, then add 50μl of standard diluent to the first and second wells, mix well; then from the first well and second
Take 100μl of each well and add them to the third and fourth wells respectively, then add 50μl of standard dilution solution to the third and fourth wells.
Mix well; then take 50μl each in the third and fourth wells and discard, then add 50μl each to the fifth and sixth wells
In the middle, add 50ul of the standard dilution solution to the fifth and sixth wells respectively, mix well;
Add 50μl to the seventh and eighth wells respectively, then add 50μl of the standard dilution solution to the seventh and eighth wells respectively, mix
After smoothing, take 50μl from the seventh and eighth holes respectively and add them to the ninth and tenth holes, then add the standards to the ninth and tenth holes respectively
50μl of the product dilution, after mixing, take 50μl from the ninth and tenth wells and discard. (After dilution, the volume of each well is 50μl,
The concentrations were 180ng / L, 120ng / L, 60ng / L, 30ng / L, 15ng / L).
2. Add sample: set blank wells separately (the blank control wells do not add samples and enzyme reagents, the rest of the steps are the same), the sample to be tested
Pinhole. Add 40μl of sample diluent to the test sample well of the enzyme-coated plate, and then add 10μl of the test sample (sample
The final dilution of the product 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
uniform.
3. Incubation: seal the plate with the sealing film and incubate at 37 ° 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 washing liquid, let stand for 30 seconds and then discard, so
Repeat 5 times and 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, and then add 50μl of developer B, mix gently, and develop color at 37 ℃ in the dark
15 minutes.
10. Termination: Add 50μl 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. Determination should be terminated
Within 15 minutes after the solution.
Calculation
Taking the concentration of the standard as the abscissa and the OD value as the ordinate, draw a standard curve on the coordinate paper, according to the sample
The corresponding concentration of OD value is found by the standard curve; multiplied by the dilution factor; or the standard concentration and OD value are used to calculate the standard
The linear regression equation of the quasi-curve, the OD value of the sample is substituted into the equation, the sample concentration is calculated, and then multiplied by the dilution factor,
This 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 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 dilute it with a certain multiple (n times) of the sample diluent 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 by the reading of the microplate reader.
6. The components of different batches of this reagent shall not be mixed.
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. Please keep the substrate away from light.
examination range:
7.5ng / L -250ng / L
Storage conditions and validity period
1. Kit storage :; 2-8 ℃.
2. Validity: 6 months
Counter Basin,Counter Top Basin,White Ceramic Basin,Above Counter Basin
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