**Chicken AIF ELISA Kit – For the Quantitative In Vitro Determination of Chicken Apoptosis-Inducing Factor in Serum, Plasma, Cerebrospinal Fluid, Tissue Homogenate, and Other Biological Fluids**
*For Laboratory Research Use Only. Not for Diagnostic or Therapeutic Procedures.*
This kit is designed for the quantitative measurement of Chicken Apoptosis-Inducing Factor (AIF) in various biological samples using a competitive ELISA method. The colorimetric detection system involves a chromogenic substrate that changes color in response to AIF levels. The intensity of the color change is measured at 450 nm using a microplate reader, and the concentration of AIF in the sample is determined by comparing it to a standard curve generated from known concentrations.
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### **Intended Use and Test Principle**
The Chicken AIF ELISA Kit is intended for research purposes only and should not be used in diagnostic or clinical settings. The assay follows a sandwich ELISA format, where AIF in the sample 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 Stop Solution halts the reaction, and the optical density (OD) is measured at 450 nm. A standard curve is created using serial dilutions of the AIF standard, allowing for accurate quantification of unknown samples.
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### **Sample Collection and Storage**
- **Serum**: Collect using a serum separator tube. Allow clotting for 2 hours at room temperature or overnight at 4°C. Centrifuge at 2000×g for 20 minutes. Store at -20°C. Avoid repeated freeze-thaw cycles.
- **Plasma**: Use heparin as an anticoagulant. Centrifuge within 30 minutes at 2000×g for 30 minutes at 2–8°C. Store at -20°C.
- **Cell Culture Supernatants, Tissue Homogenates, and Other Fluids**: Centrifuge to remove particulates. Assay immediately or store at -20°C. Ensure no hemolysis or granulation occurs.
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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 are stored at 2–8°C. Refer to the expiration date on the label.
| 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 |
**Standard Concentrations:** 480, 240, 120, 60, 30, 15 pg/mL
**Note:** If sample values exceed the highest standard, dilute with Sample Diluent and repeat the assay.
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### **Precautions**
1. Do not mix reagents from different kit lots.
2. Allow all reagents and samples 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 unused microtiter plate strips in the sealed bag with desiccant.
6. Use new pipette tips for each transfer to avoid contamination.
7. Handle all biological samples with care, as they may contain infectious agents.
8. Dispose of all waste according to local regulations.
9. Liquid waste must be treated with 1.0% sodium hypochlorite for 30 minutes before disposal.
10. Substrate solutions should be handled carefully. Discard if discolored.
11. Chromogen B contains 20% acetone; keep away from heat and 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 one month.
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### **Assay Procedure**
1. Prepare all reagents and add them to the microtiter plate.
2. Incubate for 60 minutes at 37°C.
3. Wash the plate 4 times using either manual or automated methods.
4. Add 50 µL of Chromogen A and 50 µL of Chromogen B to each well. Incubate for 15 minutes at 37°C.
5. Add 50 µL of Stop Solution to each well.
6. Read OD at 450 nm within 15 minutes.
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### **Calculation of Results**
1. Plot average OD values of standards against concentrations on a graph.
2. Subtract blank OD from all readings before plotting.
3. Determine sample concentrations by interpolating OD values on the standard curve.
4. Intra-assay and inter-assay CVs are less than 15%.
5. Assay range: 15–480 pg/mL.
6. Sensitivity: <1.0 pg/mL.
7. Cross-reactivity: No significant interference observed.
8. Storage: 2–8°C (frequent use), -20°C (long-term storage).
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### **Important Notes**
- Always follow proper lab safety protocols.
- Ensure all steps are performed accurately to maintain consistency.
- This kit is not approved for use in clinical diagnostics.
- Consult the user manual for detailed instructions and troubleshooting.
**For Research Use Only.**
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