Human ceramide ELISA kit for research use only
Human ceramide ELISA kit for experimental purposesThe kit is used for quantitative detection of human ceramide in serum, plasma, tissue, cell supernatant and related liquid samples in vitro.
Validity: 6 months
Storage conditions: 2-8 ° C
Experimental principleThe kit uses a double antibody one-step sandwich enzyme-linked immunosorbent assay (ELISA). The coated microcapsules were pre-coated with human ceramide-captured antibodies, and the specimens, standards, and HRP-labeled detection antibodies were sequentially added, 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 human ceramide in the sample. The absorbance (OD value) was measured at 450 nm using a microplate reader to calculate the sample concentration.
Human ceramide ELISA kit| Kit composition | 96-well configuration | 48-hole configuration | Remarks |
|---|---|---|---|
| Microporous plate | 8 holes × 12 | 8 holes × 6 | no |
| Standard | 0.3mL×6 tube | 0.3mL×6 tube | no |
| Sample diluent | 6mL | 3mL | no |
| Detection antibody-HRP | 10mL | 5mL | no |
| 20× washing buffer | 25mL | 15mL | Dilute according to the instructions |
| Substrate A | 6mL | 3mL | no |
| Substrate B | 6mL | 3mL | no |
| Stop solution | 6mL | 3mL | no |
| Sealing film | 2 sheets | 2 sheets | no |
| Instruction manual | 1 serving | 1 serving | no |
| Ziplock bag | 1 | 1 | no |
Remarks:
1. The concentration of the standard is: 160, 80, 40, 20, 10, 0 μmol/L
2. After a large number of normal specimen tests, the normal concentration values ​​of the specimens are within the detection range provided by the kit, and 50 μL of the sample can be directly loaded during the experiment. When part of the sample value exceeds the maximum standard concentration, the sample may be diluted with the sample dilution before the experiment.
Sample processing and requirements1. Serum: Whole blood samples should be placed at room temperature for 2 hours or 4 °C overnight, centrifuged at 1000g for 20 minutes, the supernatant can be detected, or the specimens should be stored at -20 ° C or -80 ° C, but should avoid repeated freezing melt.
2. Plasma: EDTA or heparin can be used as an anticoagulant. Centrifuge at 1000g for 2 minutes at 2 - 8 °C for 20 minutes within 30 minutes after collection, or store the specimen at -20 °C or -80 °C, but avoid repeated freezing and thawing. .
3. Cell culture supernatant or other biological specimens: centrifuge at 1000g for 20 minutes, take the supernatant to detect, or store the specimen at -20 °C or -80 °C, but avoid repeated freezing and thawing.
Note: Specimen hemolysis will affect the final test results, so hemolysis specimens should not be tested.
Reagents and equipment that are not provided1. Microplate reader (450nm)
2. High-precision sampler and tip: 0.5-10uL, 2-20uL, 20-200uL, 200-1000uL
3. 37 ° C incubator
4. Distilled or deionized water
Reagent preparationThe kit should be taken out of the refrigerated environment and should be balanced at room temperature before use.
Dilution of 20× Wash Buffer: Distilled water was diluted 1:20, ie 1 part of 20× wash buffer plus 19 parts of distilled water.
Steps1. Remove the required slats from the foil pouch after equilibrating for 20 min at room temperature. The remaining slats are 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 50 μL of the sample to be tested to the sample well; 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 (350 μL), 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) board).
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.
Precautions1. Incubate strictly in accordance with the specified time and temperature to ensure accurate results. All reagents must reach room temperature 20-25 °C before use. Store the reagents refrigerated immediately after use.
2. Incorrect cleaning can result in inaccurate results. Make sure to drain the liquid in the well as much as possible before adding the substrate. Do not let the micropores dry during the incubation.
3. Eliminate residual liquid and fingerprints on the bottom of the board, otherwise it will affect the OD value.
4. The substrate coloring solution should be colorless or very light, and the substrate liquid that has turned blue cannot be used.
5. Avoid cross-contamination of reagents and specimens to avoid erroneous results.
6. Avoid direct exposure to strong light during storage and incubation.
7. After balancing to room temperature, open the sealed bag to prevent the water droplets from condensing on the cold slats.
8. Any reagents should not be exposed to strong gases emitted by bleaching solvents or bleaching solvents. Any bleaching component will destroy the biological activity of the reagents in the kit.
9. Cannot use expired products.
10. If the disease is likely to spread, all samples should be managed and the sample and test device processed in accordance with the prescribed procedures.
Washing methodManual washing method: sucking (not touching the wall) or licking off the liquid in the microplate; laying several layers of absorbent paper on the experimental bench, and taking the microplate down slightly several times; 30 (48 times of 48T) The concentrated washing solution was diluted with distilled water 30 (20 times of 48T) and used. Repeat this process several times as needed.
Automatic washing: If there is an automatic washing machine, it should be used in the formal experiment after skilled use.
Experimental result calculationThe OD value of the tested standard is the abscissa, the concentration value of the standard is the ordinate, the standard curve is drawn on the coordinate paper or with the relevant software, and the linear regression equation is obtained, and the OD value of the sample is substituted into the equation to calculate the sample. concentration.
(This picture is for reference only)
Human ceramide ELISA kit performance1. Detection range: 5 μmol/L – 160 μmol/L.
2. Sensitivity: The minimum detection concentration is less than 1.0 μmol/L.
3. Specificity: Does not cross-react with other soluble structural analogs.
4. Repeatability: The intra-plate variation coefficient is less than 10%, and the inter-plate variation coefficient is less than 15%.
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