**Human Endoglin (ENG) ELISA Kit – For the Quantitative In Vitro Determination of Human Endoglin Concentrations in Serum, Plasma, Cerebrospinal Fluid, Tissue Homogenate, and Other Body Fluids**
**For Laboratory Research Use Only. Not for Diagnostic or Therapeutic 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 in clinical diagnostic settings. The assay is based on a competitive immunoassay principle, where the presence of Endoglin in the sample competes with a known amount of Endoglin conjugated to horseradish peroxidase (HRP) for binding sites on a pre-coated microtiter plate. After incubation, the enzyme reaction is stopped, and the color intensity is measured at 450 nm. The optical density (OD) values are then compared to a standard curve to determine the concentration of Endoglin in the samples.
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### **Intended Use and Test Principle**
This Human Endoglin (ENG) ELISA Kit is intended for laboratory research use only and is not suitable for diagnostic or therapeutic applications. The test involves a colorimetric detection method where the Stop Solution changes the color from blue to yellow. The absorbance is measured at 450 nm using a microplate reader. A set of calibration standards is included in the kit to generate a standard curve, which allows for accurate quantification of ENG concentrations in the tested samples.
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### **Sample Collection and Storage**
- **Serum**: Collect using a serum separator tube. Allow samples to clot for 2 hours at room temperature or overnight at 4°C before centrifuging at 2000×g for 20 minutes. Store aliquots at -20°C. Avoid repeated freeze-thaw cycles.
- **Plasma**: Collect using heparin as an anticoagulant. Centrifuge within 30 minutes at 2000×g for 30 minutes at 2–8°C. Store at -20°C. Avoid repeated freeze-thaw cycles.
- **Cell Culture Supernatants, Tissue Homogenates, and Other Biological Fluids**: Centrifuge to remove particulates and assay immediately or store at -20°C. Avoid repeated freezing and thawing.
**Note:** Ensure proper centrifugation, and avoid hemolysis or granules in the samples.
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### **Materials Required but Not Supplied**
1. Incubator set at 37°C
2. Microplate reader capable of measuring absorbance at 450 nm
3. Precision pipettes, disposable tips, and absorbent paper
4. Distilled or deionized water
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### **Reagents Provided**
All reagents should be stored at 2–8°C. Check the expiration date on the label before use.
| Reagent | 96 Determinations | 48 Determinations |
|--------|-------------------|-------------------|
| MicroELISA Strip Plate | 12 x 8 strips | 12 x 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 |
**Standard Concentrations:** 64, 32, 16, 8, 4, 2 ng/mL
**Note:** If sample values exceed the highest standard, dilute with Sample Diluent and repeat the assay.
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### **Precautions**
1. Do not mix reagents from different kit lots. All components are calibrated for optimal performance.
2. Allow all reagents and samples to reach room temperature (20–25°C) before use. Avoid using water baths for thawing.
3. Do not use reagents beyond their expiration date.
4. Use only deionized or distilled water for reagent dilution.
5. Keep unused strip wells in sealed pouches with desiccant at 2–8°C.
6. Use fresh pipette tips for each transfer to prevent contamination.
7. Handle all biological materials with care. Consider all blood-derived samples potentially infectious.
8. Dispose of all samples and waste according to local biosafety regulations.
9. Liquid waste must be treated with 1% sodium hypochlorite for at least 30 minutes before disposal.
10. Substrate solutions may be easily contaminated. Discard if discolored.
11. Chromogen B contains 20% acetone; keep away from heat and flame.
12. Remove all reagents from the refrigerator and allow them to warm up to room temperature before use.
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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 one month.
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### **Assay Procedure**
1. Prepare all reagents before starting the assay. It is recommended to run standards and samples in duplicate.
2. Add 50 µL of standard or sample to the appropriate wells. Blank well receives no addition.
3. Add 100 µL of HRP-conjugate reagent to all wells except the blank. Cover with adhesive strip and incubate for 60 minutes at 37°C.
4. Wash the microtiter plate 4 times.
- **Manual Washing:** Aspirate contents, fill with 1X Wash Solution, aspirate again. Repeat four times. Invert and blot dry.
- **Automated Washing:** Follow manufacturer instructions. Ensure complete aspiration and set fill volume to 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, protecting from light.
6. Add 50 µL of Stop Solution to each well. Read OD at 450 nm within 15 minutes.
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### **Calculation and Interpretation**
- Generate a standard curve by plotting average OD (450 nm) against standard concentrations.
- Subtract blank OD from all readings before interpretation.
- Use graph paper or software to construct the curve.
- Locate the OD value on the Y-axis, draw a horizontal line to the curve, and find the corresponding concentration on the X-axis.
- Each user should generate their own standard curve due to possible variations in technique, timing, and reagent age.
- Intra-assay CV < 15%, Inter-assay CV < 15%.
- Assay range: 2 ng/mL – 64 ng/mL
- Sensitivity: < 1.0 ng/mL
- Cross-reactivity: No significant cross-reactivity observed
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### **Storage and Stability**
- Store at 2–8°C for frequent use.
- For long-term storage, keep at -20°C.
- Shelf life: 6 months when stored properly.
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**Important:** Always follow safety protocols and handle all biological materials with caution. This kit is not intended for clinical use. For any questions or technical support, contact the manufacturer directly.
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