**Duck BAX ELISA Kit – For the Quantitative In Vitro Determination of Duck Bcl-2-Associated X Protein Concentrations in Serum, Plasma, Cerebrospinal Fluid, Tissue Homogenate, and Other Biological Fluids**
*For Laboratory Research Use Only. Not for Diagnostic or Therapeutic Procedures.*
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### **INTENDED USE AND TEST PRINCIPLE**
This Duck BAX ELISA Kit is specifically designed for laboratory research purposes and is not intended for use in diagnostic or therapeutic applications. The assay is based on a competitive enzyme-linked immunosorbent assay (ELISA) principle. The color change from blue to yellow occurs upon addition of the Stop Solution, and the optical density (OD) is measured at 450 nm using a microplate reader.
To determine the concentration of BAX in the sample, a set of calibration standards is included in the kit. These standards are run alongside the samples, allowing the operator to generate a standard curve of OD values versus BAX concentrations. The BAX concentration in the test samples is then calculated by comparing their OD readings to the standard curve.
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### **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. Centrifuge at 2000×g for 20 minutes. Remove the serum and analyze immediately, or aliquot and 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 freeze-thaw cycles.
- **Cell Culture Supernatants, Tissue Homogenates, and Other Biological Fluids**: Centrifuge to remove particulates, and analyze immediately or aliquot and store at -20°C. Ensure adequate centrifugation and avoid hemolysis or granulation.
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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. Precision pipettes, disposable tips, and absorbent paper
4. Distilled or deionized water
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### **REAGENTS PROVIDED**
All reagents should be stored at 2–8°C. Check the expiration date on the label before use.
| Reagent Name | 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: 100, 50, 25, 12.5, 6.25, 3.12 ng/mL.
- If sample values exceed the highest standard, dilute with Sample Diluent and repeat the test.
- Do not mix reagents from different kit lots.
- All components should be brought to room temperature (20–25°C) before use.
- Avoid using water baths for thawing.
- Never use beyond the expiration date.
- Only use deionized or distilled water for dilutions.
- Keep unused strips in sealed bags with desiccant.
- Use fresh pipette tips for each transfer to prevent cross-contamination.
- Handle all blood-derived materials with caution, as they may pose infectious risks.
- Dispose of all samples properly after inactivating any potential pathogens.
- Substrate solutions must be handled carefully; avoid exposure to heat or flame.
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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 1 month.
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### **ASSAY PROCEDURE**
1. Prepare all reagents before starting the assay.
2. Add 50 µL of standard or sample to the appropriate wells (excluding the blank well). Cover with an adhesive strip and incubate for 60 minutes at 37°C.
3. Wash the microtiter plate 4 times.
- **Manual Washing**: Aspirate the contents into a waste container, fill each well with 1X Wash Solution, and aspirate again. Repeat 4 times. Invert and blot dry.
- **Automated Washing**: Aspirate and wash four times using 1X Wash Buffer. Adjust brush to remove maximum liquid and set fill volume to 350 µL/well.
4. Add 50 µL of Chromogen Solution A and 50 µL of Chromogen Solution B to each well. Mix gently and 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.
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### **DATA ANALYSIS**
- Plot the average OD (450 nm) of each standard against its concentration on a graph.
- Calculate the mean OD for each sample and subtract the blank value.
- Locate the OD value on the Y-axis and draw a horizontal line to intersect the standard curve. From this point, draw a vertical line to the X-axis to determine the BAX concentration.
- Each user should generate their own standard curve due to possible variations in technique or environmental conditions.
- Intra-assay and inter-assay CV% are less than 15%.
- Assay range: 3.12 ng/mL – 100 ng/mL.
- Sensitivity: <1.0 ng/mL.
- Cross-reactivity: No significant cross-reactivity observed.
- Storage: 2–8°C (frequent use); -20°C for up to 6 months.
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**Important:** Always follow good laboratory practices and handle all materials with care. This kit is not intended for clinical use.
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