**Human Endostatin ELISA Kit – For the Quantitative In Vitro Determination of Human Endostatin Concentrations in Serum, Plasma, Cerebrospinal Fluid, Tissue Homogenate, and 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 Endostatin ELISA Kit is designed for laboratory research purposes only and should not be used in diagnostic procedures. The kit enables the quantitative measurement of Endostatin levels in various biological samples through an enzyme-linked immunosorbent assay (ELISA) method.
The principle of the test involves the use of a standard curve to determine Endostatin concentration. Calibration standards are included in the kit and are assayed alongside the samples. A standard curve is generated by plotting optical density (OD) values at 450 nm against known concentrations of Endostatin. The sample concentration is then determined by comparing its OD value to the standard curve.
**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. Centrifuge 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 (2–8°C). Avoid freezing and thawing.
- **Cell Culture Supernatants, Tissue Homogenates, and Other Biological Fluids**: Centrifuge to remove particulates. Assay immediately or store at -20°C. Ensure no hemolysis or granules are present.
**Materials Required (Not Supplied):**
1. Incubator 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
**Reagents Provided (Stored at 2–8°C):**
- Microtiter Strip Plate (12×8 strips / 12×4 strips)
- Standards (6 vials, 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 Solutions A & B (6.0 ml / 3.0 ml each)
- Stop Solution (6.0 ml / 3.0 ml)
- Closure Plate Membrane (2 units)
- User Manual (1 copy)
- Sealed Bags (1 unit)
**Standard Concentrations:** 100, 50, 25, 12.5, 6.25, 3.12 ng/mL. If sample values exceed the highest standard, dilute with Sample Diluent and repeat the assay.
**Precautions:**
1. Do not mix reagents from different kits. All components are matched for optimal performance.
2. Allow all reagents and materials to reach room temperature (20–25°C) before use.
3. Do not use reagents past their expiration date.
4. Store unused strips in sealed bags with desiccant.
5. Wear gloves when handling biological samples.
6. Dispose of waste after at least 30 minutes of inactivation.
7. Avoid contact with acids and sodium hypochlorite.
8. Substrate Solution must be clear; discard if blue.
9. Substrate B contains 20% acetone—keep away from heat or flame.
**Reagent Preparation:**
Dilute 1 volume of 20X Wash Solution with 19 volumes of distilled/deionized water. Store at 2–8°C for 1 month.
**Assay Procedure:**
1. Prepare all reagents. Add 50 µl of standard or sample in duplicate.
2. Add 100 µl of HRP-conjugate reagent to all wells except blank. Incubate for 60 minutes at 37°C.
3. Wash plate 4 times manually or automatically.
4. Add 50 µl of Chromogen A and B to each well. Incubate for 15 minutes at 37°C.
5. Add 50 µl Stop Solution. Color changes from blue to yellow.
6. Read OD at 450 nm. Plot standard curve and calculate sample concentration.
**Performance Characteristics:**
- Intra-assay and Inter-assay CV <15%
- Assay range: 3.12–100 ng/mL
- Sensitivity: <1.0 ng/mL
- Cross-reactivity: No significant cross-reactivity with other proteins
- Storage: 2–8°C (frequent use); 6 months at -20°C
**Note:** Always follow proper safety protocols and dispose of waste according to local regulations. For best results, each user should generate their own standard curve.
*This product is intended for research use only and is not approved for clinical diagnostics.*
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