**Human ROS ELISA Kit – For the quantitative in vitro determination of human reactive oxygen species (ROS) 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 the entire package insert carefully.
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### **INTENDED USE AND TEST PRINCIPLE**
This Human ROS ELISA Kit is designed for laboratory research purposes only and is not intended for diagnostic or therapeutic use. The kit utilizes an enzyme-linked immunosorbent assay (ELISA) to quantify reactive oxygen species (ROS) in biological samples.
The test works by measuring the optical density (OD) at 450 nm after a colorimetric reaction. A standard curve is generated by measuring known concentrations of ROS, allowing the operator to calculate the ROS levels in unknown samples based on their OD readings.
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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 repeated freezing and thawing.
- **Cell culture supernatants, tissue homogenates, and other body fluids**: Centrifuge to remove particulates. Assay immediately or store at -20°C. Avoid repeated freeze-thaw cycles.
**Note:** Ensure proper centrifugation and avoid hemolysis or presence of 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 are stored at 2–8°C. Check the expiration date on the label.
| Reagent | 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 mix reagents from different kit lots. All components are matched for optimal performance.
2. Always use the reagents provided by the manufacturer.
3. Avoid using water that is not deionized or distilled for reagent preparation.
4. Do not use any kit components past their expiration date.
5. Keep microtiter plates in sealed bags until ready for use. Store unused strips at 2–8°C with desiccant.
6. Use fresh, disposable pipette tips to prevent contamination.
7. Do not use disposable knives in the assay procedure, as no method can guarantee safety from potential infectious agents.
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### **WASTE DISPOSAL**
- **Liquid waste**: Add sodium hypochlorite to achieve a final concentration of 1.0%. Let it stand for at least 30 minutes to inactivate viruses before disposal.
- **Substrate solution**: Handle with care. If the solution appears bluish before use, discard it.
- **Chromogen B**: Contains 20% acetone. Keep away from heat and open flames.
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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 appropriate wells. Blank well receives no addition.
3. Add 100 µL of HRP-conjugate reagent to each well except the blank. Cover with an 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, and repeat four times. Invert and blot dry.
- **Automated washing**: Aspirate, wash four times, and blot dry.
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. The color will change from blue to yellow.
7. Measure OD at 450 nm within 15 minutes using a microplate reader.
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### **CALCULATION OF RESULTS**
- Plot average OD values (450 nm) against corresponding ROS concentrations to generate a standard curve.
- Subtract blank OD values from all readings before interpretation.
- Use graph paper or software to construct the curve.
- Determine the ROS concentration by locating the point of intersection on the X-axis.
**Note:** Variations in technique, timing, or kit age may affect results. Each user should generate their own standard curve.
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### **ADDITIONAL INFORMATION**
- **Intra-assay CV (%) and Inter-assay range:** 25–800 IU/mL
- **Sensitivity:** Typically less than 1.0 IU/mL
- **Cross-reactivity:** No significant cross-reactivity observed
- **Storage:** 2–8°C for frequent use; 6 months at -20°C
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Always follow safety protocols when handling biological materials and chemical reagents. Proper training and personal protective equipment (PPE) are strongly recommended.
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