**Human DAO ELISA Kit – For the quantitative in vitro determination of Human Diamine Oxidase (DAO) concentrations in serum, plasma, cerebrospinal fluid, tissue homogenate, and other body fluids. FOR LABORATORY RESEARCH USE ONLY. NOT FOR DIAGNOSTIC OR THERAPEUTIC USE.**
Before using this product, please read the entire package insert carefully.
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### **INTENDED USE AND TEST PRINCIPLE**
This Human DAO ELISA Kit is specifically designed for laboratory research purposes only and is not intended for use in diagnostic or therapeutic procedures. The assay is based on a sandwich ELISA format, where the enzyme-linked immunosorbent assay detects and quantifies DAO levels in biological samples.
The reaction involves binding of DAO to specific antibodies immobilized on a microtiter plate. A horseradish peroxidase (HRP)-conjugated secondary antibody then binds to the captured DAO, forming a complex. After adding the chromogenic substrate, a color change occurs, which is measured spectrophotometrically at 450 nm. A standard curve is generated using known concentrations of DAO, allowing the operator to determine the concentration of DAO in unknown samples by comparing their optical density (OD) values.
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### **SAMPLE COLLECTION AND STORAGE**
- **Serum**: Use a serum separator tube. Allow the sample to clot for 2 hours at room temperature or overnight at 4°C before centrifugation at 2000×g for 20 minutes. 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 of collection at 2000×g for 30 minutes at 2–8°C. Store at -20°C. Avoid repeated freezing and thawing.
- **Cell culture supernatants, tissue homogenates, and other biological fluids**: Remove particulates by centrifugation. Assay immediately or aliquot and store at -20°C. Ensure no hemolysis or granulation occurs during sample preparation.
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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. Micropipettes, 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.
| Name | 96 Determinations | 48 Determinations |
|-----------------------------|-------------------|-------------------|
| MicroELISA Stripplate | 12×8 strips | 12×4 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: 24, 12, 6, 3, 1.5, 0.75 U/mL.
- If sample results 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. Each component is calibrated for optimal performance.
2. Allow all reagents and materials to reach room temperature (20–25°C) before use. Do not use water baths to thaw samples.
3. Do not use any reagent beyond its expiration date.
4. Only use deionized or distilled water for reagent dilutions.
5. Keep unused microtiter strip wells in their sealed pouch with desiccant.
6. Use fresh pipette tips for each transfer to prevent contamination.
7. Dispose of all liquid waste properly. Add sodium hypochlorite to a final concentration of 1.0% and allow it to stand for 30 minutes before disposal.
8. Substrate solution is sensitive; if it appears blue before use, discard and prepare a fresh batch.
9. Chromogen B contains 20% acetone; keep away from heat and open flames.
10. Do not use any disposable knives that may have come into contact with rat blood due to potential infectious agents.
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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 all wells except the blank. Cover with adhesive strip and incubate for 60 minutes at 37°C.
4. Wash the microtiter plate 4 times:
- **Manual Washing:** Aspirate plate contents, fill with 1X Wash Solution, aspirate again. Repeat 4 times.
- **Automated Washing:** Aspirate, wash 4 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 shift from blue to yellow. Read OD at 450 nm within 15 minutes.
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### **CALCULATION AND INTERPRETATION**
1. Plot the average OD values (450 nm) against the corresponding standard concentrations to generate a standard curve.
2. Subtract the blank OD value from all measurements before interpreting results.
3. 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 sample concentration.
4. Variability in results can occur due to differences in pipetting, washing, incubation time, or kit age. Each user should establish their own standard curve.
5. Intra-assay CV: <10%. Inter-assay range: 0.75 pg/mL – 24 pg/mL.
6. Sensitivity: Typically less than 0.1 U/mL.
7. Cross-reactivity: No significant cross-reactivity observed.
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### **STORAGE AND STABILITY**
- Store at 2–8°C for frequent use.
- For long-term storage, keep at -20°C.
- Shelf life: 6 months when stored properly.
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**Please read all instructions carefully and follow safety guidelines. This kit is for research use only.**
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