**Human DKK3 ELISA Kit – For the quantitative in vitro determination of Human Dickkopf 3 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 DKK3 ELISA Kit is designed for laboratory research purposes only and is not intended for diagnostic or therapeutic procedures. The kit employs a sandwich ELISA technique to quantitatively measure DKK3 levels in various biological samples. The reaction involves binding of DKK3 to specific antibodies coated on the microtiter plate. After incubation and washing steps, a chromogenic substrate is added, producing a color change that is proportional to the concentration of DKK3. The optical density (OD) is measured at 450 nm, and results are calculated by comparing sample OD values to a standard curve generated from known concentrations.
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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 centrifuging 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**: Centrifuge to remove particulates. Assay immediately or aliquot and store at -20°C. Ensure no hemolysis or contamination occurs during 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. 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 expiration date on label.
| Reagent Name | 96 Determinations | 48 Determinations |
|--------------|-------------------|-------------------|
| MicroELISA Stripplate | 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: 200, 100, 50, 25, 12.5, 6.25 ng/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 kits. All components are calibrated for optimal performance.
2. Allow all reagents and materials to reach room temperature (20–25°C) before use. Do not use water baths for thawing.
3. Do not use reagents past their expiration date.
4. Only use deionized or distilled water for dilution.
5. Keep microtiter plates in sealed bags until needed. Unused strips should be stored at 2–8°C with desiccant.
6. Use fresh pipette tips for each transfer to avoid cross-contamination.
7. Handle all biological samples with care, as they may contain infectious agents.
8. Dispose of all samples following proper biohazard protocols.
9. Liquid waste must be treated with sodium hypochlorite (1.0% final concentration) for at least 30 minutes before disposal.
10. Substrate solutions are sensitive; discard if discolored or contaminated.
11. Chromogen B contains 20% acetone—keep away from heat and flame.
12. Allow all reagents to warm to 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 beginning.
2. Add 100 µL of standards, samples, and blanks to the appropriate wells. Cover with adhesive strips and incubate for 60 minutes at 37°C.
3. Wash the plate 4 times using either manual or automated methods:
- **Manual Washing**: Aspirate and fill each well with 1X Wash Solution, then aspirate again. Repeat four times.
- **Automated Washing**: Aspirate and wash four times, ensuring 350 µL per well per wash.
4. After washing, 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.
5. Add 50 µL of Stop Solution to each well. The color should turn yellow. If green or uneven, gently tap the plate.
6. Measure OD at 450 nm within 15 minutes using a microplate reader.
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### **CALCULATION OF RESULTS**
1. Plot average OD values (450 nm) of standards against their concentrations to generate a standard curve.
2. Subtract blank OD values from all measurements before interpretation.
3. Use graph paper or software to construct the curve.
4. Locate the sample OD on the Y-axis and draw a horizontal line to intersect the curve. Then draw a vertical line to the X-axis to determine DKK3 concentration.
5. Intra-assay and inter-assay CVs are less than 15%.
6. Assay range: 6.25 ng/mL to 200 ng/mL.
7. Sensitivity: <1.0 ng/mL.
8. Cross-reactivity: No significant cross-reaction with other proteins.
9. Storage: 2–8°C for frequent use; -20°C for long-term storage (up to 6 months).
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**Important Note:** Always follow good laboratory practices when handling biological samples and reagents. This kit is not suitable for clinical diagnosis. For any questions or technical support, contact the manufacturer directly.
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