Human Apoptosis Related Factor Ligand (FASL) ELISA Kit Instructions
2025-07-13 11:10:17
**Human Apoptosis-Related Factor Ligand (FASL) ELISA Kit**
*For the quantitative in vitro determination of Human FASL concentrations in serum, plasma, cerebrospinal fluid, tissue homogenate, and other body fluids.*
**Intended for laboratory research use only. Not for diagnostic or therapeutic purposes.**
**INTENDED USE AND TEST PRINCIPLE**
This FASL ELISA kit is designed for laboratory research and is not intended for use in diagnostic or clinical procedures. The assay is based on a sandwich immunoassay principle, where FASL 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 reaction is stopped with a stop solution, and the optical density (OD) is measured at 450 nm using a microplate reader.
**CALIBRATION AND QUANTIFICATION**
The kit includes a set of standard solutions with known concentrations of FASL (2000, 1000, 500, 250, 125, and 62.5 pg/ml). These standards are run alongside the samples to generate a standard curve. The OD values of the unknown samples are compared against this curve to determine their FASL concentration.
**SAMPLE COLLECTION AND STORAGE**
- **Serum**: Use 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**: Collect using 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 biological fluids**: Centrifuge to remove particulates. Assay immediately or store at -20°C. Avoid freeze-thaw cycles.
- **Note**: Ensure adequate centrifugation and avoid hemolysis or granulation in samples.
**MATERIALS REQUIRED BUT NOT SUPPLIED**
- 37°C incubator
- Microplate reader capable of measuring absorbance at 450 nm
- Precision pipettes, disposable tips, and absorbent paper
- Distilled or deionized water
**REAGENTS PROVIDED**
All reagents should be stored at 2–8°C. Check the expiration date on the label.
- MicroELISA Strip Plate (12×8 or 12×4 strips)
- Standard (6 vials, 0.5 ml/vial)
- Sample Diluent (6.0 ml or 3.0 ml)
- HRP-Conjugate Reagent (10.0 ml or 5.0 ml)
- 20X Wash Solution (25 ml or 15 ml)
- Chromogen Solution A (6.0 ml or 3.0 ml)
- Chromogen Solution B (6.0 ml or 3.0 ml)
- Stop Solution (6.0 ml or 3.0 ml)
- Closure Plate Membrane (2 units)
- User Manual (1 copy)
- Sealed Bags (1 unit)
**PRECAUTIONS**
- Do not mix reagents from different kits.
- Allow all reagents and samples to reach room temperature before use.
- Do not use beyond the expiration date.
- Only use deionized or distilled water for dilutions.
- Store unused strips in sealed bags with desiccant.
- Wear gloves during handling; treat all biological materials as potentially infectious.
- Dispose of waste properly after inactivation.
- Avoid exposure to heat or flame when handling Chromogen B (contains 20% acetone).
**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 and standards. Run each sample and standard in duplicate.
2. Add 50 µl of standard or sample to appropriate wells.
3. Add 100 µl of HRP-conjugate reagent to all wells except the blank. Cover with adhesive strip and incubate for 60 minutes at 37°C.
4. Wash the plate four 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, protecting from light.
6. Add 50 µl of Stop Solution to each well. Mix gently if color change is uneven.
7. Measure OD at 450 nm within 15 minutes.
**CALCULATION OF RESULTS**
- Plot average OD values of standards vs. concentration.
- Subtract blank OD from all measurements.
- Determine unknown concentrations by comparing OD to the standard curve.
- Intra-assay and inter-assay CVs are <15%.
- Sensitivity: <10 pg/ml.
- Cross-reactivity: No significant cross-reaction with other proteins.
- Storage: 2–8°C (frequent use), or -20°C for long-term storage (up to 6 months).
**NOTES**
- Always read the entire package insert before use.
- Follow good laboratory practices to ensure accuracy and safety.
- This kit is not suitable for clinical diagnosis.
**FOR RESEARCH USE ONLY.**
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