**Human DcR3 ELISA Kit – For the Quantitative In Vitro Determination of Human Soluble Decoy Receptor (DcR3) in Serum, Plasma, Cerebrospinal Fluid, Tissue Homogenate, and Other Biological Fluids**
*For Laboratory Research Use Only. Not Intended for Diagnostic or Therapeutic Procedures.*
This ELISA kit is designed for the quantitative determination of human soluble Decoy Receptor 3 (DcR3) in various biological samples. The assay is based on the enzyme-linked immunosorbent assay (ELISA) principle, utilizing a sandwich format to measure DcR3 levels. The results are obtained by comparing the optical density (OD) values of the samples against a standard curve generated from known concentrations of DcR3.
**Intended Use & Test Principle**
The Human DcR3 ELISA Kit is intended for laboratory research purposes only and should not be used in diagnostic or therapeutic applications. The test involves incubating standards and samples in microtiter wells coated with specific antibodies against DcR3. After washing, a horseradish peroxidase (HRP)-conjugated secondary antibody is added, followed by a chromogenic substrate. The reaction is stopped, and the OD is measured at 450 nm. A standard curve is plotted, and sample concentrations are determined by interpolation.
**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 centrifugation at 2000×g for 20 minutes. Remove serum and 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 for 30 minutes at 2–8°C. Store at -20°C. Avoid freeze-thaw cycles.
- **Cell Culture Supernatants, Tissue Homogenates, and Other Biological Fluids**: Centrifuge to remove particulates and assay immediately or store at -20°C. Ensure no hemolysis or granules are present.
**Materials Required (Not Supplied)**
1. Incubator set at 37°C
2. Microplate reader capable of measuring absorbance at 450 nm
3. Precision pipettes, disposable tips, and absorbent paper
4. Distilled or deionized water
**Reagents Provided**
All reagents are stored at 2–8°C. Refer to the expiration date on the label.
- Microplate: 12×8 strips (96 determinations), 12×4 strips (48 determinations)
- 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 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 units
- User Manual: 1 unit
- Sealed Bags: 1 unit
**Precautions**
1. Do not mix reagents from different kit lots. Standards, conjugates, and plates are matched for optimal performance.
2. Allow all reagents and samples to reach room temperature (20–25°C) before use. Do not thaw using water baths.
3. Do not use reagents past their expiration date.
4. Use only deionized or distilled water for dilutions.
5. Keep microtiter plates in sealed bags until use. Unused strips should be stored at 2–8°C with desiccant.
6. Use fresh pipette tips for each transfer to prevent cross-contamination.
7. All blood-derived products should be treated as potentially infectious. Follow good laboratory practices.
8. Dispose of all samples and reagents following proper biohazard protocols. Allow liquid to stand for at least 30 minutes to inactivate viruses before disposal.
9. Substrate solutions can be easily contaminated. Avoid exposure to heat or flame.
**Reagent Preparation & 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.
**Assay Procedure**
1. Prepare all reagents before starting the assay.
2. Add 50 µL of standard or sample to appropriate wells (excluding blank). Cover with adhesive strip and incubate for 60 minutes at 37°C.
3. Wash the microtiter plate 4 times manually or automatically.
4. 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. Read OD at 450 nm using a microplate reader.
6. Plot the average OD values of the standards against their concentrations to generate a standard curve. Determine sample concentrations by comparing their OD values to the curve.
**Interpretation of Results**
- Calculate the mean OD for each standard and sample. Subtract the blank value from all readings.
- Construct the standard curve using graph paper or software. 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 the concentration.
- Intra-assay and inter-assay CV% are less than 15%.
- Assay range: 3.75 pg/mL – 120 pg/mL.
- Sensitivity: <1.0 pg/mL.
- Cross-reactivity: No significant cross-reactivity observed.
**Storage**
- Store unused kits at 2–8°C for frequent use, or at -20°C for long-term storage (up to 6 months).
**Note:** If sample values exceed the highest standard, dilute with Sample Diluent and repeat the assay. Always read the entire package insert before use.
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