**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 product, please read this entire package insert carefully. This ELISA kit is designed for research purposes only and should not be used in diagnostic or clinical settings.
**INTENDED USE AND TEST PRINCIPLE**
The Human DKK1 ELISA Kit is intended for laboratory research use only. It utilizes a sandwich immunoassay format to quantitatively determine the concentration of DKK1 in biological samples. The color change from blue to yellow, induced by the Stop Solution, is proportional to the DKK1 concentration. Standards are run simultaneously with samples to generate a standard curve, allowing for accurate quantification of DKK1 levels in unknown samples.
**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 (2–8°C). Store at -20°C. Avoid repeated freezing.
- **Cell Culture Supernatant, Tissue Homogenate, and Other Biological Fluids**: Centrifuge to remove particulates. Assay immediately or store at -20°C. Ensure no hemolysis or contamination occurs.
**MATERIALS REQUIRED BUT NOT SUPPLIED**
1. 37°C incubator
2. Microplate reader capable of measuring absorbance at 450 nm
3. Pipettes, disposable tips, and absorbent paper
4. Distilled or deionized water
**REAGENTS PROVIDED**
All reagents are stored at 2–8°C. Check expiration date on label.
- MicroELISA Strip Plate: 12x8 strips / 12x4 strips
- Standard (6 vials): 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 A: 6.0 ml / 3.0 ml
- Chromogen 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
**NOTES**
1. Standard concentrations: 160, 80, 40, 20, 10, 5 ng/mL.
2. If sample values exceed the highest standard, dilute with Sample Diluent and repeat the assay.
3. Bring all reagents to room temperature (20–25°C) before use. Do not thaw using water baths.
4. Do not use beyond the expiration date.
5. Use only deionized or distilled water for reagent dilution.
6. Keep unused strips in their pouch with desiccant at 2–8°C.
7. Use fresh pipette tips for each transfer to avoid cross-contamination.
8. Do not use disposable knives for testing, as no method guarantees safety from infectious agents.
9. Liquid waste must be treated with sodium hypochlorite (final concentration 1.0%) and left for 30 minutes before disposal.
10. Substrate A should not be used if it appears bluish.
11. Substrate B contains 20% acetone—keep away from heat and flame.
12. Allow all reagents to reach room temperature before use.
**REAGENT PREPARATION AND STORAGE**
- **Wash Solution (1X)**: Dilute 1 volume of 20X Wash Solution with 19 volumes of deionized water. Store at 2–8°C for up to 1 month.
**ASSAY PROCEDURE**
1. Prepare all reagents before starting.
2. Add 100 µL of standards, samples, and blanks to microtiter plate. Cover with adhesive strip and incubate for 60 minutes at 37°C.
3. Wash plate 4 times (manual or automated).
- **Manual Washing**: Aspirate, fill with 1X Wash Solution, aspirate again. Repeat 4 times. Invert and blot dry.
- **Automated Washing**: Aspirate, wash 4 times with 350 µL/well. Blot dry.
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.
**CALCULATION**
Plot average OD values (450 nm) against standard concentrations. Subtract blank OD from all readings. Use graph paper or software to construct the standard curve. Interpolate sample concentrations from the curve.
**PERFORMANCE CHARACTERISTICS**
- Sensitivity: <1.0 ng/mL
- Range: 5–160 ng/mL
- Intra-assay CV: <10%
- Inter-assay CV: <15%
- Cross-reactivity: No significant cross-reactivity observed.
**STORAGE**
- Store at 2–8°C for frequent use.
- For long-term storage, keep at -20°C for up to 6 months.
**WARNING**
This kit is for research use only. Handle with care and follow all safety guidelines. Always wear appropriate personal protective equipment.
**Please refer to the complete user manual for detailed instructions and troubleshooting.**
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