**Human Endostatin ELISA Kit – For the quantitative in vitro determination of Human Endostatin concentrations in serum, plasma, cerebrospinal fluid, tissue homogenate, and other body fluids. FOR LABORATORY RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.**
Before using this product, please read this package insert carefully. This Endostatin 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 Endostatin levels in biological samples.
The kit includes a set of calibration standards that are run alongside the test samples. These standards help generate a standard curve by plotting optical density (OD) values against known Endostatin concentrations. Once the standard curve is established, the OD readings from the test samples are compared to determine their Endostatin concentration.
**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 centrifugation at 2000 ×g for 20 minutes. Remove serum and assay immediately or aliquot and store 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 freeze-thaw cycles.
- **Cell culture supernatants, tissue homogenates, and other biological fluids:** Centrifuge to remove particulates. Assay immediately or store at -20°C. Ensure no hemolysis or debris is present.
**Materials Required (Not Supplied):**
- 37°C incubator
- Microplate reader capable of measuring absorbance at 450 nm
- Precision pipettes, disposable tips, and absorbent paper
- Distilled or deionized water
**Reagents Provided (Stored at 2–8°C):**
- Microtiter strip plate (12×8 strips or 12×4 strips)
- Standard (6 vials, 0.5 ml/vial)
- Sample Diluent (6.0 ml or 3.0 ml)
- HRP-Conjugate Reagent (10.0 ml or 5.0 ml)
- 20X Wash Solution (25 ml or 15 ml)
- Chromogen Solution A (6.0 ml or 3.0 ml)
- Chromogen Solution B (6.0 ml or 3.0 ml)
- Stop Solution (6.0 ml or 3.0 ml)
- Closure Plate Membrane (2 units)
- User Manual (1 copy)
- Sealed bags (1 unit)
**Note:** Standard concentrations are 100, 50, 25, 12.5, 6.25, and 3.12 ng/mL. If sample values exceed the highest standard, dilute with Sample Diluent and repeat the assay.
**Precautions:**
- Do not mix reagents from different kits. All components are calibrated for optimal performance.
- Allow all reagents to reach room temperature (20–25°C) before use. Avoid using water baths for thawing.
- Do not use reagents beyond the expiration date.
- Store unused strips in their sealed pouch with desiccant.
- Wear gloves during the procedure. Handle all biological materials as potentially hazardous.
- Inactivate waste for 30 minutes before disposal.
- Avoid contact with acids and sodium hypochlorite.
- Substrate solution should not be blue; discard if it appears bluish.
- Chromogen B contains 20% acetone—keep away from heat and flame.
**Reagent Preparation:**
- Wash Solution (1X): Dilute 1 volume of 20X Wash Solution with 19 volumes of distilled or deionized water. Store at 2–8°C for up to one month.
**Assay Procedure:**
1. Prepare all reagents before starting. 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 the blank. Cover with adhesive strip and incubate for 60 minutes at 37°C.
4. Wash microtiter plate 4 times manually or automatically.
5. Add 50 μL of Chromogen A and 50 μL of Chromogen B to each well. Incubate for 15 minutes at 37°C, protecting from light.
6. Add 50 μL of Stop Solution to each well. Color changes from blue to yellow. Gently tap if color change is uneven.
7. Read OD at 450 nm. Subtract blank OD from all values.
**Data Interpretation:**
- Plot average OD values of standards vs. concentration to create a standard curve.
- Locate the OD value of each sample on the Y-axis and draw a horizontal line to intersect the curve. From there, draw a vertical line to the X-axis to determine the concentration.
**Performance Characteristics:**
- Intra-assay and Inter-assay CV < 15%
- Assay range: 3.12 ng/mL – 100 ng/mL
- Sensitivity: < 1.0 ng/mL
- Cross-reactivity: No significant cross-reactivity with other proteins
**Storage:**
- 2–8°C for frequent use; 6 months at -20°C
**Important Notes:**
- Always follow safety protocols when handling biological samples.
- Each user should generate their own standard curve due to possible variations in technique or environmental factors.
- This kit is intended for research use only. Not approved for clinical diagnosis.
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