**Human ROS ELISA Kit – For the Quantitative In Vitro Determination of Human Reactive Oxygen Species 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 this entire package insert carefully. This ELISA kit is designed for research purposes only and should not be used in clinical diagnostic settings.
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### **INTENDED USE AND TEST PRINCIPLE**
The Human ROS ELISA Kit is intended for laboratory research use only. It allows for the quantitative determination of reactive oxygen species (ROS) in various biological samples such as serum, plasma, cerebrospinal fluid, tissue homogenates, and other body fluids. The test is based on an enzyme-linked immunosorbent assay (ELISA) principle.
A stop solution is added to terminate the reaction, causing a color change from blue to yellow. The optical density (OD) values of the standards are measured simultaneously with the samples, allowing the creation of a standard curve. The ROS concentration in the samples is then calculated by comparing their OD values to the 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. 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. Store at -20°C. Avoid freeze-thaw cycles.
- **Cell Culture Supernatants, Tissue Homogenates, and Other Biological Fluids**: Remove particulates by centrifugation. Assay immediately or store at -20°C. Avoid repeated freezing and thawing.
**Note:** Ensure proper centrifugation and avoid hemolysis or granulation 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 Name | 96 Determinations | 48 Determinations |
|--------------|-------------------|-------------------|
| MicroELISA Stripplate | 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: 800, 400, 200, 100, 50, 25 IU/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. All components are matched for optimal performance.
2. Do not use reagents beyond their expiration date.
3. Use only deionized or distilled water for diluting reagents.
4. Do not mix disposable pipette tips.
5. Keep all kit reagents refrigerated until use.
6. Allow reagents to reach room temperature (20–25°C) before use.
7. Always use fresh disposable tips to prevent contamination.
8. Substrate B contains 20% acetone; keep it away from heat and flame.
9. Handle liquid waste with care. Add sodium hypochlorite to a final concentration of 1.0% and let stand for 30 minutes before disposal.
10. The substrate solution is sensitive to contamination. If it appears bluish, do not 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. Add standards and samples in duplicate to the microtiter plate.
2. Add 50 μL of standard or sample to each well (blank well receives nothing).
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 plate 4 times manually or automatically. After washing, invert and blot dry.
5. Add 50 μL of Chromogen Solution A and 50 μL of Chromogen Solution 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 changes from blue to yellow. Measure OD at 450 nm within 15 minutes.
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### **CALCULATION AND INTERPRETATION**
- Plot the average OD (450 nm) of each standard against its concentration to generate a standard curve.
- Subtract the blank OD value from all readings before interpretation.
- Use graph paper or software to construct the curve.
- At the point of intersection, draw a vertical line to the X-axis to determine the ROS concentration.
- Intra-assay CV (%) and inter-assay range: 25–800 IU/mL.
- Sensitivity: <1.0 IU/mL.
- No cross-reactivity or interference observed.
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### **STORAGE AND STABILITY**
- Store at 2–8°C for frequent use; for long-term storage, keep at -20°C for up to six months.
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**This kit is intended for research use only. Do not use in diagnostic procedures.**
**Always follow safety guidelines and proper disposal methods.**
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