Detection principle
The kit uses a double antibody one-step sandwich enzyme-linked immunosorbent assay (ELISA).
Pre-package
Be
Vitreinogen (VTG)
The coated antibody is coated with microcapsules, and the test sample, standard, and HRP-labeled detection antibody are sequentially added, and the cells are washed 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 depth of the color and the sample
Vitreinogen (VTG)
Present
Positive correlation. The absorbance (OD value) was measured at a wavelength of 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 to avoid any cell irritation during the operation. 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: anticoagulation with EDTA, citrate or heparin. 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 added to the appropriate amount of physiological saline and mashed. The supernatant was taken by centrifugation at 3000 rpm for 10 minutes.
5. Preservation: If the sample is not detected in time after collection, please pack it once and freeze it at -20 °C to 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
Operational precautions
1.
The kit was 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.
Incubation is carried out in strict accordance with the time indicated in the instructions, the amount of liquid added and the order.
5.
Shake well all liquid components before use.
Kit composition
Note: Standard (S0-S5) concentration
In turn
for:
0, 30, 60, 120, 240, 480 ng/mL
Reagent preparation
Dilution of 20× Wash Buffer: 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 350 μL of washing solution into each well, soak for 1 min, and wash the plate 5 times.
Steps
1.
The required slats were taken out from the aluminum foil pouch after equilibrating for 20 min at room temperature, and the remaining slats were sealed back to 4 ° C with a ziplock bag.
2.
Set standard and sample holes
, standard wells are added with different concentrations of standard 50μL;
3.
Sample hole first
Sample to be tested
10μL, then
Add sample diluent
4
0 μL;
Blank holes are not added.
4.
Except for blank holes,
100 μL of horseradish peroxidase (HRP)-labeled detection antibody was added to each well of the standard well and the sample well, and the reaction well was sealed with a sealing plate, and incubated at 37 ° C in a water bath or incubator for 60 min.
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.
50 μL of each of the substrates A and B was added to each well, and incubated at 37 ° C for 15 min in the dark.
7.
50 μL of the stop solution was added to each well, and the OD value of each well was measured 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 linear regression coefficient of the standard and the expected concentration correlation coefficient R value, greater than or equal to 0.9900.
2.
Sensitivity: the minimum detection concentration is less than
1.0ng/mL
.
3.
Specificity: Does not cross-react with other soluble structural analogs.
4.
Repeatability: The coefficient of variation between the plates and the plates 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 in clinical trials or
people
Physical experiments, otherwise all the consequences will be borne by the experimenter, the company is not responsible.
2.
In strict accordance with the instructions, the experimenter violates the instructions, and the consequences are borne by the experimenter.
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