**Human DKK4 ELISA Kit – For the Quantitative In Vitro Determination of Human Dickkopf 4 Concentrations in Serum, Plasma, Cerebrospinal Fluid, Tissue Homogenate, and Other Body Fluids**
**For Laboratory Research Use Only. Not for Diagnostic or Therapeutic Procedures.**
Before using this product, please read the entire package insert carefully. This ELISA kit is designed for research purposes only and is not intended for use in diagnostic procedures.
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### **INTENDED USE AND TEST PRINCIPLE**
This Human DKK4 ELISA Kit is intended for laboratory research use only. It utilizes a sandwich ELISA technique to quantitatively measure the concentration of DKK4 in various biological samples. The test works by binding DKK4 to specific antibodies immobilized on a microtiter plate. A secondary HRP-conjugated antibody then binds to the captured DKK4, and a chromogenic substrate produces a color change that is proportional to the amount of DKK4 present. The optical density (OD) is measured at 450 nm, and the concentration of DKK4 in the sample is determined by comparing it 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 at 2000×g at 2–8°C. Store at -20°C. Avoid repeated freezing.
- **Cell Culture Supernatants, Tissue Homogenates, and Other Biological Fluids**: Remove particulates by centrifugation. Assay immediately or store at -20°C. Ensure no hemolysis or granules are present 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. Pipettes, disposable tips, and absorbent paper
4. Distilled or deionized water
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### **REAGENTS PROVIDED**
| Reagent Name | 96 Determinations | 48 Determinations |
|----------------------------------|-------------------|-------------------|
| MicroELISA Strip Plate | 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 |
*Standard concentrations: 80, 40, 20, 10, 5, 2.5 ng/mL.*
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### **PRECAUTIONS AND INSTRUCTIONS**
1. All reagents should be brought to room temperature (20–25°C) before use. Do not thaw using water baths.
2. Do not use any kit components beyond their expiration date.
3. Only use deionized or distilled water for diluting reagents.
4. Keep unused strip wells in their sealed pouch with desiccant at 2–8°C.
5. Use fresh pipette tips for each transfer to prevent cross-contamination.
6. Do not use disposable knives or tools that may introduce contamination.
7. Liquid waste must be treated with sodium hypochlorite (final concentration 1.0%) and left for at least 30 minutes to inactivate viruses.
8. Substrate solutions should be checked for discoloration before use. If blue, discard.
9. Substrate B contains 20% acetone; keep away from heat or open flame.
10. Ensure all reagents are at room temperature before starting the assay.
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### **REAGENT PREPARATION**
- **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 beginning.
2. Add 100 µL of standards, samples, and blanks to the microtiter plate. Cover with adhesive strips and incubate for 60 minutes at 37°C.
3. Wash the plate 4 times using either manual or automated washing methods.
4. Add 50 µL of Chromogen A and 50 µL of Chromogen B to each well. Incubate for 15 minutes at 37°C, protected from light.
5. Add 50 µL of Stop Solution to each well. Read OD at 450 nm within 15 minutes.
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### **CALCULATION AND INTERPRETATION**
- Plot the average OD values of the standards against their concentrations.
- Subtract the blank OD value from all measurements.
- Construct a standard curve using graph paper or statistical software.
- Determine the DKK4 concentration by interpolating the sample OD on the standard curve.
- Intra-assay CV: < 10%, Inter-assay range: 2.5–80 ng/mL.
- Sensitivity: < 1.0 ng/mL.
- Cross-reactivity: No significant cross-reactivity observed with other proteins.
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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.
- Do not expose to extreme temperatures or humidity.
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**Please follow all instructions carefully to ensure accurate and reliable results.**
**This product is not intended for human diagnostic use.**
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