**Duck P53 ELISA Kit – For the Quantitative In Vitro Determination of Duck P53 Protein Concentrations in Serum, Plasma, Cerebrospinal Fluid, Tissue Homogenate, and Other Biological Fluids**
*For Laboratory Research Use Only. Not for Diagnostic or Therapeutic Procedures.*
Before using this product, please read the entire package insert carefully. This Duck P53 ELISA Kit is designed for laboratory research purposes only and is not intended for use in diagnostic or clinical procedures. The assay is based on the enzyme-linked immunosorbent assay (ELISA) principle, which allows for the quantitative measurement of P53 protein levels in various biological samples.
The test works by detecting the presence of Duck P53 in the sample through a colorimetric reaction. A stop solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm. Standard curves are generated by measuring known concentrations of P53, allowing for accurate quantification of unknown samples. The optical density (OD) values obtained from the samples are compared to the standard curve to determine their P53 concentration.
**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 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 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 contamination occurs.
**Materials Required but 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 (Stored at 2–8°C)**
- Microtiter Strip Plate: 12×8 strips (96 determinations), 12×4 strips (48 determinations)
- Standards (6 vials, 0.5 mL/vial)
- Sample Diluent: 6.0 mL (96), 3.0 mL (48)
- HRP-Conjugate Reagent: 10.0 mL (96), 5.0 mL (48)
- 20X Wash Solution: 25 mL (96), 15 mL (48)
- Chromogen Solution A: 6.0 mL (96), 3.0 mL (48)
- Chromogen Solution B: 6.0 mL (96), 3.0 mL (48)
- Stop Solution: 6.0 mL (96), 3.0 mL (48)
- Closure Plate Membrane: 2 units
- User Manual: 1 unit
- Sealed Bags: 1 unit
**Standard Concentrations**: 500, 250, 125, 62.5, 31.2, 15.6 pg/mL
**Note**: If sample values exceed the highest standard, dilute with Sample Diluent and repeat the assay.
**Precautions**
1. Do not mix reagents from different kit lots.
2. Allow all reagents to reach room temperature (20–25°C) before use.
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. Store unused strips with desiccant.
6. Use fresh pipette tips for each transfer to prevent cross-contamination.
7. Handle all biological samples with care, as they may contain infectious agents.
8. Dispose of waste properly after inactivation for at least 30 minutes.
9. Substrate solutions must be used before discoloration occurs.
10. Chromogen B contains 20% acetone; keep away from heat or flame.
**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.
**Assay Procedure**
1. Prepare all reagents before starting.
2. Add 100 μL of standards, samples, and controls to the microtiter plate.
3. Cover with adhesive strips 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 in the dark.
6. Add 50 μL of Stop Solution to each well. The color should turn yellow.
7. Measure OD at 450 nm within 15 minutes.
**Data Interpretation**
- Plot average OD values against standard concentrations to create a standard curve.
- Subtract blank well OD from all readings.
- Use graph paper or software to generate the curve.
- Locate the OD value on the Y-axis and find the corresponding P53 concentration on the X-axis.
**Performance Characteristics**
- Intra-assay and Inter-assay CV% < 15%
- Assay Range: 15.6 pg/mL – 500 pg/mL
- Sensitivity: <10 pg/mL
- Cross-reactivity: No significant interference observed
- Storage: 2–8°C (frequent use); -20°C (up to 6 months)
**Important Notes**
- Always follow good laboratory practices when handling biological materials.
- Ensure all steps are performed accurately to maintain consistency and reliability.
- Regularly check equipment calibration and reagent integrity.
- Maintain proper documentation for all experimental procedures.
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