This kit can only be used for scientific research and should not be used for medical diagnosis.
Human apelin 36 (AP36) ELISA test kit instruction manual
Detection principle
The kit uses a double antibody one-step sandwich enzyme-linked immunosorbent assay (ELISA). The coated microcapsules pre-coated with apelin 36 (AP36) antibody were sequentially added to the specimen, standard, and HRP-labeled detection antibody, and incubated and thoroughly washed. Using the substrate TMB to develop color, TMB is converted to blue under the catalysis of peroxidase and converted to the final yellow color by the action of an acid. The color depth is positively correlated with apelin 36 (AP36) in the sample. The absorbance (OD value) was measured at 450 nm using a microplate reader to calculate the sample concentration.
Sample collection, processing and storage methods
1. Serum: Use a tube containing no pyrogen and endotoxin. Avoid any cell irritation during the procedure. After collecting the blood, centrifuge and centrifuge for 10 minutes at 3000 rpm to quickly and carefully separate the serum and red blood cells.
2. Plasma: EDTA, citrate or heparin anticoagulation. The supernatant was taken by centrifugation at 3000 rpm for 30 minutes.
3. Cell supernatant: Centrifuge at 3000 rpm for 10 minutes to remove particles and polymer.
4. Tissue homogenization: The tissue is mashed by adding appropriate amount of physiological saline. The supernatant was taken by centrifugation at 3000 rpm for 10 minutes.
5. Storage: If the sample is not detected in time after collection, please dispense it once, freeze it at -20 °C, avoid repeated freezing and thawing, thaw at room temperature and ensure that the sample is fully thawed evenly.
Bring your own items
1. Microplate reader (450nm)
2. High-precision sampler and tip: 0.5-10uL, 2-20uL, 20-200uL, 200-1000uL
3.37 °C incubator operation precautions 1. The kit is stored at 2-8 ° C, and equilibrated for 20 minutes at room temperature before use. The concentrated washing liquid taken out from the refrigerator will crystallize, which is a normal phenomenon, and the water bath is heated to completely dissolve the crystals before use.
2. The slats not used in the experiment should be immediately put back into the ziplock bag and sealed (low temperature dry) for storage.
3. The S0 standard with a concentration of 0 can be regarded as a negative control or blank; the sample has been diluted 5 times according to the instructions, and the final result multiplied by 5 is the actual concentration of the sample.
4. Incubate the operation strictly in accordance with the time indicated in the instructions, the amount of liquid added, and the sequence.
5. Shake well all liquid components before use.
Kit composition
Name 96-well configuration 48-well configuration Remarks Microporous microplates 12 wells × 8 12 wells × 4 No standard 0.3 mL * 6 tubes 0.3 mL * 6 tubes No sample dilution 6 mL 3 mL No detection antibody - HRP 10 mL 5 mL None 20×washing buffer 25mL 15mL Diluted substrate A 6mL 3mL without substrate B 6mL 3mL without stop solution 6mL 3mL without sealing film 2 sheets 2 sheets without instructions 1 part 1 part without zipper bag 1 1 no note : The concentration of standard (S0-S5) is: 0, 0.5, 1, 2, 4, 8 ng/mL
Preparation of reagents 20× Wash buffer dilution: Distilled water was diluted 1:20, ie 1 part of 20× wash buffer plus 19 parts of distilled water.
Washing method
1. Manually wash the plate: Drain the liquid in the hole, fill each hole with the washing liquid, let stand for 1 min, then drain the liquid in the hole, pat dry on the absorbent paper, and wash the plate 5 times. 2. Automatic washing machine: Inject 350μL of washing solution into each hole, soak for 1min, and wash the plate 5 times. Procedure 1. Remove the required slats from the foil pouch after equilibrating for 20 min at room temperature. The remaining slats were sealed back to 4 °C with a ziplock bag.
2. Set standard and sample wells, and add standard concentration of 50μL to each standard.
3. Add 10 μL of the sample to the sample and add 40 μL of the sample dilution; blank holes are not added.
4. In addition to the blank wells, add 100 μL of horseradish peroxidase (HRP)-labeled detection antibody to each well of the standard wells and sample wells, seal the wells with a sealing membrane, and incubate in a 37 ° C water bath or incubator. 60min.
5. Discard the liquid, pat dry on the absorbent paper, fill each well with the washing solution, let stand for 1 min, remove the washing solution, pat dry on the absorbent paper, and repeat the washing 5 times (can also be washed with a washing machine).
6. Add 50 μL of substrate A and B to each well and incubate at 37 ° C for 15 min in the dark. 7. Add 50 μL of stop solution to each well, and measure the OD value of each well at a wavelength of 450 nm within 15 min.
Result judgment
Draw a standard curve: In the Excel worksheet, the standard concentration is used as the abscissa, and the OD value is plotted as the ordinate. The linear regression curve of the standard is drawn, and the concentration values ​​of each sample are calculated according to the curve equation.
Kit performance
1. Accuracy: The R value of the linear regression of the standard and the expected concentration is greater than or equal to 0.9900.
2. Sensitivity: The minimum detection concentration is less than 0.1 ng/mL.
3. Specificity: Does not cross-react with other soluble structural analogs.
4. Repeatability: The coefficient of variation between the plate and the plate is less than 15%.
5. Storage: 2-8 ° C, protected from light and moisture.
6. Validity: 6 months
Disclaimer
1. The kit is for research use only and should not be used for clinical trials or human experiments. Otherwise, the consequences will be borne by the experimenter and the company will not be responsible. 2. Operate in strict accordance with the instructions, the experimenter violates the instructions, and the consequences are borne by the experimenter.
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