**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 USE IN DIAGNOSTIC PROCEDURES.**
Before using this product, please read the entire package insert carefully. This ELISA kit is designed for research purposes only and should not be used in diagnostic or therapeutic settings.
**INTENDED USE AND TEST PRINCIPLE**
The Duck P53 ELISA Kit is intended for laboratory research use. The test is based on the principle of a sandwich ELISA, where the P53 antigen binds to specific antibodies coated on the microtiter plate. A horseradish peroxidase (HRP)-conjugated secondary antibody is then added, followed by a chromogenic substrate that produces a color change. The intensity of the color is directly proportional to the concentration of P53 in the sample. The optical density (OD) is measured at 450 nm, and the P53 concentration is determined by comparing the sample OD values with a standard curve.
**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. 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 body fluids**: Centrifuge to remove particulates. Assay immediately or store at -20°C. Avoid repeated freeze-thaw cycles.
*Note: Ensure proper centrifugation and avoid hemolysis or granules in samples.*
**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 (Store all reagents at 2–8°C)**
| Reagent | 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 | 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 500, 250, 125, 62.5, 31.2, and 15.6 pg/mL. 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. All components are calibrated for optimal performance.
2. Allow all reagents and samples to reach room temperature (20–25°C) before use. Do not thaw in a water bath.
3. Do not use reagents past their expiration date.
4. Only use deionized or distilled water for dilutions.
5. Keep unused strip wells in their sealed pouch with desiccant.
6. Use fresh pipette tips for each transfer to prevent cross-contamination.
7. Treat all biological samples as potentially infectious. Follow good lab practices.
8. Dispose of all waste properly, ensuring viral inactivation for at least 30 minutes before disposal.
9. Substrate solutions must be handled carefully. If discolored, do not use.
10. Chromogen B contains 20% acetone—keep away from heat and flames.
**REAGENT PREPARATION**
- **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 blanks to the microtiter plate. Cover with adhesive strips and incubate for 60 minutes at 37°C.
3. Wash the plate 4 times using either manual or automated washing.
- *Manual*: Aspirate, fill with wash solution, aspirate again. Repeat 4 times. Invert and blot dry.
- *Automated*: Aspirate and wash 4 times, adjust brush to remove maximum liquid, fill 350 μL/well.
4. Add 50 μL of Chromogen A and 50 μL of Chromogen B to each well. Mix gently and incubate for 15 minutes at 37°C, protected from light.
5. Add 50 μL of Stop Solution to each well. The color will change from blue to yellow. If uneven, gently tap the plate.
6. Read OD at 450 nm within 15 minutes. Subtract blank OD from all values.
**RESULT ANALYSIS**
- Plot average OD values (450 nm) against standard concentrations.
- Determine sample concentrations by comparing OD values to the standard curve.
- Each user should generate their own standard curve.
- Intra-assay CV <15%, Inter-assay CV <15%.
- Assay range: 15.6 pg/mL to 500 pg/mL.
- Sensitivity: <10 pg/mL.
- Cross-reactivity: No significant cross-reactivity observed.
**STORAGE**
- Store at 2–8°C for frequent use; for long-term storage, keep at -20°C. Shelf life: 6 months when stored properly.
**NOTE:** Always follow safety protocols and ensure proper handling of all biological materials. This kit is not intended for clinical use.
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