**Human DKK1 ELISA Kit – For the Quantitative In Vitro Determination of Human Dickkopf 1 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 kit, please read the entire package insert carefully. This ELISA kit is designed for research purposes only and is not intended for use in clinical diagnostics.
### **INTENDED USE AND TEST PRINCIPLE**
The Human DKK1 ELISA Kit is a competitive immunoassay used to quantitatively measure the concentration of Human Dickkopf-1 (DKK1) in various biological samples. The assay is based on the principle of enzyme-linked immunosorbent assay (ELISA), where specific antibodies bind to DKK1 in the sample. A colorimetric reaction occurs, and the optical density (OD) at 450 nm is measured. By comparing the OD values of the test samples with those of a standard curve, the concentration of DKK1 can be accurately determined.
### **SAMPLE COLLECTION AND STORAGE**
- **Serum**: Collect using a serum separator tube. Allow samples to clot for 2 hours at room temperature or overnight at 4°C. Centrifuge at 2000×g for 20 minutes. Assay immediately or store 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 freeze-thaw cycles.
- **Cell Culture Supernatants, Tissue Homogenates, and Other Biological Fluids**: Centrifuge to remove particulates. Assay immediately or store at -20°C. Ensure no hemolysis or contamination.
### **MATERIALS REQUIRED BUT NOT SUPPLIED**
1. Incubator 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
### **REAGENTS PROVIDED**
| Reagent Name | 96 Determinations | 48 Determinations |
|---------------------------|-------------------|-------------------|
| MicroELISA Strip Plate | 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: 160, 80, 40, 20, 10, 5 ng/mL
- If sample values exceed the highest standard, dilute with Sample Diluent and repeat the assay.
### **PREPARATION AND STORAGE OF REAGENTS**
- **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.
### **ASSAY PROCEDURE**
1. Prepare all reagents before starting the assay.
2. Add 100 µL of standards, samples, and blanks to the microtiter plate.
3. Cover with an adhesive strip and incubate for 60 minutes at 37°C.
4. Wash the plate 4 times using either manual or automated washing methods.
- *Manual Washing:* Aspirate and fill each well with 1X Wash Solution, repeating 4 times.
- *Automated Washing:* Aspirate and wash four times, adjusting
Brush and fill volume accordingly.
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, protecting from light.
6. Add 50 µL of Stop Solution to each well. The color changes from blue to yellow.
7. Read OD at 450 nm within 15 minutes using a microplate reader.
### **CALCULATION AND INTERPRETATION**
- Plot the average OD values (450 nm) against the corresponding DKK1 concentrations to generate a standard curve.
- Subtract the blank OD value from all other 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 DKK1 concentration in the samples.
### **PERFORMANCE CHARACTERISTICS**
- **Intra-assay CV (%):** < 10%
- **Inter-assay CV (%):** < 15%
- **Sensitivity:** < 1.0 ng/mL
- **Cross-reactivity:** No significant cross-reactivity observed
- **Storage:** 2–8°C (for frequent use); -20°C for long-term storage (up to 6 months)
### **SAFETY AND DISPOSAL**
- **Liquid Waste:** Add sodium hypochlorite to achieve a final concentration of 1.0%. Let stand for at least 30 minutes before disposal.
- **Substrate Solution A/B:** Avoid exposure to light. Do not use if discolored.
- **Substrate B:** Contains 20% acetone. Keep away from heat and flame.
- **General Safety:** Handle all reagents with care. Wear appropriate PPE. Avoid contact with skin and eyes.
### **NOTES**
- Always allow reagents and samples to reach room temperature (20–25°C) before use.
- Do not thaw reagents in a water bath.
- Do not use any kit components beyond their expiration date.
- Use only deionized or distilled water for dilutions.
- Discard unused strips in sealed bags with desiccant.
- Use fresh pipette tips for each transfer to prevent cross-contamination.
This kit provides a reliable and accurate method for measuring DKK1 levels in various biological matrices. Proper handling and adherence to the protocol are essential for consistent and reproducible results.
Cellulose Sponge Pad
Puyang Maikefu Commodity Co., LTD , https://www.maikefufoam.com