This kit can only be used for scientific research, not for medical diagnostics (Human) Endothelin 1 (ET-1) ELISA test kit instructions for use Principles of detection The kit uses double antibody one-step sandwich enzyme-linked immunosorbent assay (ELISA ). To the coated microwells pre-coated with ET-1 antibody, add the specimen, standard, and
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The detection antibody labeled with HRP is incubated and washed thoroughly. Color development with substrate TMB, TMB
It is converted into blue under the catalysis of peroxidase, and into the final yellow under the action of acid. The color depth is positively correlated with endothelin 1 (ET-1) in the sample. The absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm, and the sample concentration was calculated.
Sample collection, processing and storage methods
1. Serum: Use a test tube that does not contain pyrogens and endotoxins. Avoid any cell irritation during the operation. After collecting blood, centrifuge at 3000 rpm for 10 minutes to quickly and carefully separate serum and red blood cells.
2. Plasma: EDTA, citrate or heparin anticoagulation. Take the supernatant by centrifugation at 3000 rpm for 30 minutes.
3. Cell supernatant: centrifuge at 3000 rpm for 10 minutes to remove particles and polymers.
4. Tissue homogenate: Add tissue to the right amount of saline and mash. Take the supernatant by centrifugation at 3000 rpm for 10 minutes.
5. Preservation: If the samples are not tested in time after collection, please aliquot according to the dosage and freeze in
-20 ℃, avoid repeated freezing and thawing, thaw at room temperature and ensure that the sample is thawed evenly and fully.
Bring your own items
1. Microplate reader (450nm)
2. High-precision sampler and tip: 0.5-10uL, 2-20uL, 20-200uL, 200-1000uL
3. Precautions for operation of 37 ℃ thermostat
1. Store the kit at 2-8 ° C and equilibrate at room temperature for 20 minutes before use. The concentrated washing liquid taken out of the refrigerator will have crystals, which is a normal phenomenon. The water bath is heated to completely dissolve the crystals before use.
2. The slats not used in the experiment should be immediately returned to the ziplock bag, sealed (dry at low temperature) and stored.
3. S0 standard with a concentration of 0 can be regarded as a negative control or blank; the sample after pretreatment does not need to be diluted, just take 50μL and add it.
4. Perform the incubation operation strictly in accordance with the time, amount of liquid and sequence indicated in the manual.
5. Shake all liquid components thoroughly before use.
Kit composition name 96-well configuration 48-well configuration Remarks Microwell microplate 12 well × 8 strips 12 wells × 4 strips No standard 0.3mL * 6 tubes 0.3mL * 6 tubes without biotin 1mL 0.5mL No detection antibody-HRP 10mL 5mL None
20 × Washing buffer 25mL 15mL Dilute according to the instructions Substrate A 6mL 3mL No substrate B 6mL 3mL No stop solution 6mL 3mL Unsealed plate film 2 sheets 2 sheets without instructions 1 part 1 part without ziplock bag 1 piece 1 piece without injection : The concentration of standard products (S0-S5) is: 0, 10, 20, 40, 80, 160 pg / mL
Reagent preparation
Dilution of 20 × washing buffer: 1:20 dilution of distilled water, that is, 1 part of 20 × washing buffer plus 19 parts of distilled water.
Washing method
1. Manually wash the plate: throw away the liquid in the hole, fill each hole with the washing liquid, leave the hole in the hole for 1min, shake off the liquid in the hole, pat dry on absorbent paper, and wash the plate 5 times in this way.
2. Automatic plate washing machine: Inject 350μL of washing solution into each well, soak for 1min, wash the plate 5 times.
Steps
1. Take out the required slats from the aluminum foil bag after equilibrating at room temperature for 20min. The remaining slats are sealed with a self-sealing bag and put back at 4 ℃.
2. Set up standard wells and sample wells, add 50μL of standard products of different concentrations to the standard wells;
3. Add 10μL of biotin to the sample well, then add 50μL of the sample to be tested;
4. Then add 100μL of detection antibody labeled with horseradish peroxidase (HRP) to each well of the standard well and the sample well, seal the reaction well with a sealing film, and incubate for 60min in a 37 ° C water bath or thermostat.
5. Discard the liquid, pat dry on absorbent paper, fill each well with washing liquid, let stand for 1min, shake off the washing liquid, pat dry on absorbent paper, and repeat washing the plate 5 times (you can also wash the plate with a washing machine).
6. Add 50 μL of substrate A and B to each well, and incubate at 37 ° C in the dark for 15 minutes.
7. Add 50μL of stop solution to each well, and measure the OD value of each well at 450nm within 15min.
Result judgment to draw a standard curve: In the Excel worksheet, the standard concentration is used as the abscissa, corresponding
The OD value is used as the ordinate, a linear regression curve of the standard product is drawn, and the concentration value of each sample is calculated according to the curve equation.
Kit performance
1. Accuracy: the correlation coefficient R between the linear regression of the standard product and the expected concentration is greater than or equal to
0.9900.
2. Sensitivity: The minimum detection concentration is less than 1.0 pg / mL.
3. Specificity: Does not cross-react with other soluble structural analogs.
4. Repeatability: The coefficients of variation within and between panels are less than 15%.
5. Storage: 2-8 ℃, 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 or human experiments, otherwise all the consequences will be borne by the experimenter, and the company will not be responsible.
2. Strictly follow the instructions, and the experimenter violates the instructions, and the consequences will be borne by the experimenter.
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