**Human Decorin ELISA Kit – For the quantitative in vitro determination of Human Decorin concentrations in serum, plasma, cerebrospinal fluid, tissue homogenate, and other body fluids. Intended for laboratory research use only. NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES.**
Before using this kit, please read the entire package insert carefully. This ELISA kit is designed for research purposes only and should not be used for diagnostic or clinical applications.
The principle of this assay is based on a sandwich ELISA format. The color change from blue to yellow occurs when the Stop Solution is added, and the optical density (OD) is measured at 450 nm. A standard curve is generated by plotting OD values against known Decorin concentrations. Sample concentrations are then determined by comparing their OD readings to this standard curve.
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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 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). Store at -20°C. Avoid repeated freezing and thawing.
- **Cell culture supernatants, tissue homogenates, and other biological fluids:** Centrifuge to remove particulates. Assay immediately or aliquot and store at -20°C. Ensure no hemolysis or granules are present in the samples.
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**Materials Required (Not Supplied):**
1. 37°C incubator
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 (Stored at 2–8°C):**
| Reagent Name | 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 |
| 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 are 200, 100, 50, 25, 12.5, and 6.25 pg/mL. 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. Standards, conjugates, and plates are matched for optimal performance.
2. Allow all reagents and materials to reach room temperature (20–25°C) before use. Avoid using water baths to thaw.
3. Do not use reagents beyond their expiration date.
4. Only use deionized or distilled water for dilution.
5. Keep microtiter plates in the sealed bag until needed. Unused strips should be stored with desiccant at 2–8°C.
6. Use fresh disposable pipette tips for each transfer to prevent contamination.
7. Dispose of all blood-derived products as potentially infectious. Follow proper biosafety protocols.
8. Inactivate viruses in liquid waste by adding 1% sodium hypochlorite and letting it sit for 30 minutes before disposal.
9. Substrate solution is sensitive to contamination. Chromogen B contains 20% acetone—keep away from heat or flame.
10. Let all reagents reach 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 starting. 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 each well.
4. Wash the plate four times with 1X Wash Solution. After the final wash, invert and blot dry.
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. The color should turn yellow. If green or uneven, gently tap the plate.
7. Measure OD at 450 nm using a microplate reader.
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**Data Interpretation:**
- Plot average OD values (450 nm) of standards vs. concentration.
- Subtract blank OD from all samples.
- Locate the sample OD on the Y-axis and draw a horizontal line to intersect the standard curve. Draw a vertical line to the X-axis to determine concentration.
- Intra-assay and inter-assay CVs are <15%.
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**Performance Characteristics:**
- **Assay Range:** 6.25 pg/mL to 200 pg/mL
- **Sensitivity:** <1.0 pg/mL
- **Cross-reactivity:** No significant cross-reactivity observed with other proteins
- **Storage:** 2–8°C (frequent use); -20°C (long-term storage, up to 6 months)
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**Important Notes:**
- Always follow safety guidelines when handling biological samples.
- This product is not intended for diagnostic use.
- Read and understand the entire protocol before performing the test.
**For Research Use Only.**
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