**Chicken AIF ELISA Kit – For the Quantitative In Vitro Determination of Chicken Apoptosis-Inducing Factor (AIF) Concentrations in Serum, Plasma, Cerebrospinal Fluid, Tissue Homogenate, and Other Body Fluids**
*For Laboratory Research Use Only. Not for Diagnostic or Therapeutic Procedures.*
This ELISA kit is designed for the quantitative detection of Apoptosis-Inducing Factor (AIF) in various biological samples. The assay is based on a sandwich immunoassay principle using specific antibodies. The reaction is terminated by a stop solution, which changes the color from blue to yellow. The optical density (OD) is measured at 450 nm with a microplate reader. Calibration standards are included to generate a standard curve, allowing for accurate quantification of AIF levels in unknown samples.
**Intended Use**
The Chicken AIF ELISA Kit is intended for laboratory research use only and is not suitable for diagnostic or therapeutic applications.
**Test Principle**
The kit employs a competitive or sandwich ELISA method to detect AIF. Standards and samples are incubated in microtiter wells coated with specific antibodies. After washing, a horseradish peroxidase (HRP)-conjugated secondary antibody is added, followed by a chromogenic substrate. The reaction is stopped, and the resulting color intensity is measured at 450 nm. The concentration of AIF in the sample is determined by comparing its OD value to the standard curve.
**Sample Collection and Storage**
- **Serum**: Use serum separator tubes. 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**: Collect using heparin as an anticoagulant. Centrifuge within 30 minutes at 2000×g (2–8°C). Store at -20°C.
- **Tissue Homogenates, Cell Culture Supernatants, and Other Biological Fluids**: Centrifuge to remove particulates. 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**
All reagents are stored at 2–8°C. Check expiration date on the label.
| 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 |
| 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.
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 and materials to reach room temperature (20–25°C) before use.
3. Do not use expired reagents.
4. Use only deionized or distilled water for dilutions.
5. Keep unused strip plates in sealed bags with desiccant.
6. Use fresh pipette tips for each transfer to avoid cross-contamination.
7. Wear gloves during the procedure; treat all biological samples as potentially infectious.
8. Dispose of all waste properly. For liquid waste, add sodium hypochlorite to 1.0% final concentration and let sit for 30 minutes.
9. Substrate solutions must be used before they turn bluish.
10. Chromogen B contains 20% acetone; keep away from heat or flame.
11. Let all reagents reach room temperature before starting the assay.
**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. Add standards and samples to the microtiter plate. Cover with adhesive strips and incubate for 60 minutes at 37°C.
2. Wash the plate 4 times manually or automatically.
3. 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.
4. Add 50 µL of Stop Solution to each well. Mix gently if color change is uneven.
5. Measure OD at 450 nm within 15 minutes.
**Calculation of Results**
1. Plot average OD values (450 nm) against standard concentrations.
2. Subtract blank OD from all measurements.
3. Determine AIF concentration by matching sample OD to the standard curve.
4. Each user should generate their own standard curve.
5. Intra-assay and inter-assay CVs are <15%.
6. Assay range: 15–480 pg/mL.
7. Sensitivity: <1.0 pg/mL.
8. No significant cross-reactivity observed.
9. Storage: 2–8°C (frequent use); 6 months at -20°C.
**Note**: This product is intended for research purposes only. Always follow proper safety protocols when handling biological samples.
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