Human Apoptosis Related Factor Ligand (FASL) ELISA Kit Instructions

**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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