**Human Endoglin (ENG) ELISA Kit – For the Quantitative In Vitro Determination of Human Endoglin Concentrations in Serum, Plasma, Cerebrospinal Fluid, Tissue Homogenate, and Body Fluids**
**For Laboratory Research Use Only. NOT FOR USE IN DIAGNOSTIC PROCEDURES.**
Before using this product, please read the entire package insert carefully. This ELISA kit is designed for research purposes only and should not be used for diagnostic or therapeutic applications. The assay is based on the enzyme-linked immunosorbent assay (ELISA) principle, which allows for the accurate quantification of endoglin levels in biological samples.
The Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm using a spectrophotometer. To determine the concentration of ENG in your samples, the kit includes a set of calibration standards. These standards are run alongside your samples, allowing you to generate a standard curve that correlates optical density (OD) with ENG concentration. By comparing the OD values of your samples to this curve, you can accurately calculate their ENG levels.
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**Sample Collection and Storage**
- **Serum**: Use a serum separator tube. Allow samples to clot for 2 hours at room temperature or overnight at 4°C before centrifuging for 20 minutes at approximately 2000×g. Store samples at -20°C immediately after aliquoting. Avoid repeated freeze-thaw cycles.
- **Plasma**: Collect plasma using heparin as an anticoagulant. Centrifuge within 30 minutes of collection at 2000×g for 30 minutes at 2–8°C. Store at -20°C and avoid freeze-thaw cycles.
- **Cell Culture Supernatants, Tissue Homogenates, and Other Biological Fluids**: Remove particulates by centrifugation and either assay immediately or store at -20°C. Avoid repeated freeze-thaw cycles.
- **Note**: Ensure proper centrifugation and avoid hemolysis or granules in the samples.
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**Materials Required but Not Supplied**
1. 37°C incubator
2. Microplate reader capable of measuring absorbance at 450 nm
3. Precision pipettes, disposable pipette tips, and absorbent paper
4. Distilled or deionized water
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**Reagents Provided (Stored at 2–8°C)**
| Reagent | 96 Determinations | 48 Determinations |
|--------|------------------|-------------------|
| MicroELISA Strip Plate | 12×8 strips | 12×4 strips |
| Standard (6 vials, 0.5 ml/vial) | 0.5 ml/vial | 0.5 ml/vial |
| Sample Diluent | 6.0 ml | 3.0 ml |
| HRP-Conjugate Reagent | 10.0 ml | 5.0 ml |
| 20X Wash Solution | 25 ml | 15 ml |
| Chromogen Solution A | 6.0 ml | 3.0 ml |
| Chromogen Solution B | 6.0 ml | 3.0 ml |
| Stop Solution | 6.0 ml | 3.0 ml |
| Closure Plate Membrane | 2 | 2 |
| User Manual | 1 | 1 |
| Sealed Bags | 1 | 1 |
**Note**: Standard concentrations: 64, 32, 16, 8, 4, 2 ng/mL. If sample values exceed the highest standard, dilute with Sample Diluent and repeat the assay.
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**Precautions**
1. Do not substitute reagents between different kit lots. Standards, conjugates, and plates are matched for optimal performance.
2. Allow all reagents and materials to reach room temperature (20–25°C) before use. Do not use water baths for thawing.
3. Do not use reagents beyond their expiration date.
4. Use only deionized or distilled water for dilution.
5. Do not remove microtiter plate strips from the storage bag until needed. Store unused strips in their pouch with desiccant.
6. Use fresh pipette tips for each transfer to prevent contamination.
7. Avoid using acid-dissolvable knives. No known method guarantees complete safety from infectious agents in rat-derived products. Treat all blood derivatives as potentially infectious.
8. Dispose of all samples properly to inactivate viruses.
9. Liquid waste: Add sodium hypochlorite to a final concentration of 1.0% and let stand for 30 minutes before disposal.
10. Substrate solution is easily contaminated. If it appears bluish, do not use.
11. Chromogen B contains 20% acetone—keep away from heat or flame.
12. Bring all reagents to room temperature before starting the assay.
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**Reagent Preparation and Storage**
- **Wash Solution (1X)**: Dilute 1 volume of 20X Wash Solution with 19 volumes of deionized or distilled water. Store at 2–8°C for up to 1 month.
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**Assay Procedure**
1. Prepare all reagents before beginning. Run standards and samples in duplicate.
2. Add 50 µL of standard or sample to appropriate wells. Blank well receives no addition.
3. Add 100 µL of HRP-conjugate reagent to all wells except blank. Cover with adhesive strip and incubate for 60 minutes at 37°C.
4. Wash microtiter plate 4 times.
- **Manual Washing**: Aspirate contents, fill with 1X Wash Solution, aspirate again. Repeat four times. Invert and blot dry.
- **Automated Washing**: Aspirate and wash four times with 1X Wash Solution. Fill 350 µL/well.
5. Add 50 µL of Chromogen A and 50 µL of Chromogen B to each well. Mix gently and incubate for 15 minutes at 37°C, protected from light.
6. Add 50 µL of Stop Solution to each well. The color will change from blue to yellow. Read OD at 450 nm within 15 minutes.
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**Calculation**
1. Plot average OD (450 nm) of standards vs. concentration on a graph.
2. Subtract blank OD from all readings before interpretation.
3. Use graph paper or software to construct the standard curve.
4. Locate the OD value of the sample on the Y-axis, draw a horizontal line to the curve, then a vertical line to the X-axis to find the corresponding concentration.
5. Each user should create their own standard curve.
6. Intra-assay and inter-assay CVs are <15%.
7. Assay range: 2–64 ng/mL.
8. Sensitivity: <1.0 ng/mL.
9. Cross-reactivity: No significant cross-reactivity observed.
10. Storage: 2–8°C (frequent use); 6 months at -20°C.
**Please read the full instructions before use.**
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