**Human DKK3 ELISA Kit – For the Quantitative In Vitro Determination of Human Dickkopf 3 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 this entire package insert carefully. This ELISA kit is designed for research purposes only and is not intended for diagnostic or clinical procedures. The assay is based on a sandwich immunoassay principle, where the optical density (OD) values of the standards are measured at 450 nm to generate a standard curve. Sample concentrations are then determined by comparing their OD values to the standard curve.
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### **INTENDED USE AND TEST PRINCIPLE**
This DKK3 ELISA Kit is specifically designed for the quantitative determination of Human Dickkopf-3 (DKK3) in various biological samples such as serum, plasma, cerebrospinal fluid, tissue homogenates, and other body fluids. It is suitable for use in laboratory research settings only. The test involves binding of DKK3 to immobilized antibodies, followed by detection with a HRP-conjugated secondary antibody. A chromogenic substrate is added, producing a color change that is measured spectrophotometrically.
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### **SAMPLE COLLECTION AND STORAGE**
**Serum:**
Use serum separator tubes. Allow samples to clot for 2 hours at room temperature or overnight at 4°C before centrifugation 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 of collection at 2000×g for 30 minutes at 2–8°C. Store at -20°C. Avoid multiple 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 granulation 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 (Storage: 2–8°C)**
| Reagent | 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 |
**Note:** Standard concentrations are 200, 100, 50, 25, 12.5, and 6.25 ng/mL. If sample values exceed the highest standard, dilute with Sample Diluent and re-run the assay.
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### **PRECAUTIONS**
1. Do not mix reagents from different kit lots. All components are calibrated for optimal performance.
2. Allow all reagents and materials to reach room temperature (20–25°C) before use. Avoid using water baths for thawing.
3. Do not use reagents past their expiration date.
4. Use only deionized or distilled water for reagent dilutions.
5. Keep microtiter plates sealed until needed. Store unused strips with desiccant at 2–8°C.
6. Use fresh pipette tips for each transfer to prevent cross-contamination.
7. Handle all biological samples with care, following biosafety protocols.
8. Dispose of waste properly. For liquid waste, add sodium hypochlorite to 1.0% concentration and let sit for 30 minutes before disposal.
9. Avoid contamination of substrate solutions. Discard if they appear blue before use.
10. Chromogen B contains 20% acetone—keep away from heat and flame.
11. Let all reagents reach 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 one month.
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### **ASSAY PROCEDURE**
1. Prepare all reagents before starting.
2. Add 100 µL of standards, samples, and blank to the microtiter plate.
3. Cover the plate with an adhesive strip and incubate for 60 minutes at 37°C.
4. Wash the plate 4 times manually or automatically.
5. 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.
6. Add 50 µL of Stop Solution to each well. The color should turn yellow. If not uniform, gently tap the plate.
7. Measure OD at 450 nm within 15 minutes using a microplate reader.
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### **CALCULATION OF RESULTS**
1. Plot the average OD values of the standards on the Y-axis against their concentrations on the X-axis.
2. Calculate the mean OD for each standard and sample. Subtract the blank OD from all readings.
3. Use graph paper or software to construct the standard curve.
4. Locate the OD value of the sample on the Y-axis, draw a horizontal line to the curve, then a vertical line to the X-axis to determine the concentration.
5. Each user should generate their own standard curve due to possible variations in technique.
6. Intra-assay and inter-assay CV% are <15%.
7. Assay range: 6.25 ng/mL to 200 ng/mL.
8. Sensitivity: <1.0 ng/mL.
9. Cross-reactivity: No significant cross-reactivity with other proteins.
10. Storage: 2–8°C (frequent use); -20°C (long-term storage).
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**Important:** Always follow safety guidelines and proper waste disposal procedures. This kit is not intended for use in diagnostic or clinical applications.
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