Enzyme-linked immunoassay for human anti-Mullerian hormone (AMH)
Kit instruction manual
Read this manual carefully before use. This ELISA kit is based on the principle of biotin double antibody sandwich technology to detect human anti-Mullerian hormone (AMH), which can only be used for research purposes, not for medical diagnosis.
Uses: Used for the determination of anti-Mullerian hormone (AMH) in human serum, plasma and related liquid samples.
working principle
This kit uses biotin double antibody sandwich enzyme-linked immunosorbent assay (ELISA) to determine the level of human anti-Mullerian hormone (AMH) in the sample. Add anti-Müllerian hormone (AMH) to the enzyme-labeled wells pre-coated with human anti-Müllerian hormone (AMH) monoclonal antibody; after incubation, add biotin-labeled anti-AMH antibody, and then Streptavidin-HRP combines to form an immune complex, which is then incubated and washed to remove unbound enzymes, and then substrates A and B are added to produce a blue color, which is converted into a final yellow color under the action of acid . The color depth is positively correlated with the concentration of human anti-Mullerian hormone (AMH) in the sample.
Kit composition
1
Standard product (16ng / ml)
0.5ml
7
Developer A liquid
6ml
2
Standard dilution
3ml
8
Developer B liquid
6ml
3
Enzyme coated plate
12 holes × 8
9
Stop solution
6ml
4
Streptavidin-HRP
6ml
10
Instructions
1 serving
5
30 times concentrated washing solution
20ml
11
Sealing film
2 sheets
6
Biotin-labeled anti-AMH antibody
1ml
12
sealed bag
1
Reagents and equipment needed but not provided
1. 37 ℃ thermostat.
2. Standard specification microplate reader.
3. Precision pipettes and disposable tips
4. Distilled water,
5. Disposable test tube
6. Absorbent paper
Precautions
1. The kit taken from 2-8 ° C should be equilibrated at room temperature for at least 30 minutes before opening the kit. If the enzyme-coated plates are not used up after opening, the slats should be stored in sealed bags.
2. The sampler should be used at each step of sample addition, and the accuracy should be regularly checked to avoid test errors.
3. Strictly follow the instructions, and the test results must be determined based on the reading of the microplate reader.
4. To avoid cross-contamination, avoid repeated use of the tip and sealing film in your hand.
5. Unused other reagents should be packed or covered. Do not mix reagents of different batches. Use before warranty.
6. Substrate B is sensitive to light and avoid prolonged exposure to light.
Washing method
Manual plate washing method: throw away the liquid in the enzyme label plate; place a few layers of absorbent paper on the experimental table, the enzyme label plate is forced to pat several times downward; inject at least 0.35ml of the diluted washing solution into the well and soak 2 minutes. Repeat this process several times as needed.
Automatic plate washing: If there is an automatic plate washing machine, it should be used in the formal experiment process after being used skillfully.
Specimen requirements
1. The samples containing NaN3 cannot be detected because NaN3 inhibits horseradish peroxidase (HRP) activity.
2. Specimens are extracted as soon as possible after collection, and extraction is performed according to relevant literature, and experiments should be conducted as soon as possible after extraction. If the test cannot be performed immediately, the specimen can be stored at -20 ℃, but repeated freezing and thawing should be avoided
Operating procedures
1. Dilution of standard products: (This kit provides one original standard product, please follow the instructions to dilute it in a small test tube):
8ng / ml
(No. 5 standard product)
120μl of original standard is added to 120μl of standard dilution
4ng / ml
(Standard 4)
120μl No. 5 standard is added to 120μl standard dilution
2ng / ml
(Standard No. 3)
120μl No. 4 standard is added to 120μl standard dilution
1ng / ml
(Standard No. 2)
Add 120μl of standard No. 3 to 120μl of standard dilution
0.5ng / ml
(No. 1 standard product)
120μl No. 2 standard is added to 120μl standard dilution
2. The number of slats required is determined by the number of samples to be tested plus the number of standard products. It is recommended to make multiple holes for each standard and blank hole. Each sample is determined according to its own quantity, and those that can use multiple holes can be used for multiple holes.
3. Sample addition: 1) blank well: no sample is added to the blank control well, biotin-labeled anti-AMH antibody, streptavidin-HRP, only the developer A & B and the stop solution are added, and the remaining steps are the same; 2) standard Pinhole: add 50μl of standard, 50μl of streptomycin-HRP (the biotin antibody has been integrated in the standard beforehand, so it is not added); 3) Sample well to be tested: add 40μl of sample, then add 10μl of anti-AMH antibody 50 μl of streptavidin-HRP was covered with a sealing membrane, gently shaken and mixed, and incubated at 37 ° C for 60 minutes.
4. Solution: Dilute the 30-fold concentrated washing solution with distilled water 30-fold and set aside.
5. Washing: Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with the washing solution, let it stand for 30 seconds, then discard, repeat 5 times and pat dry.
6. Color development: Add 50μl of developer A to each well, then add 50μl of developer B, mix gently, and develop for 10 minutes in the dark at 37 ℃.
7. Stop: add 50μl of stop solution to each well to stop the reaction (at this time, the blue color turns to yellow).
8. Measurement: The absorbance (OD value) of each well was measured in sequence with the blank air conditioner at zero and 450 nm wavelength. The measurement should be performed within 10 minutes after adding the stop solution.
9. Calculate the linear regression equation of the standard curve according to the concentration of the standard product and the corresponding OD value, and then calculate the corresponding sample concentration on the regression equation according to the OD value of the sample. It can also be calculated using various application software.
Summary of operating procedures:
Prepare reagents, samples and standards
Add prepared samples and standards, biotin-labeled secondary antibody and enzyme-labeled reagent, and react at 37 ℃ for 60 minutes
Wash the plate 5 times, add color developing solutions A and B, and develop at 37 ℃ for 10 minutes
Add stop solution
Read OD value within 10 minutes
Calculation
Detection range: 0.05ng / ml → 15ng / ml.
Specification: 96T / box
Storage: 2-8 ℃
Validity: 6 months (2-8 ℃).
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