Human ENA-78 ELISA Kit

**Human ENA-78 ELISA Kit – For Quantitative In Vitro Determination of Human Epithelial Neutrophil Activating Peptide 78 (ENA-78) in Serum, Plasma, Cerebrospinal Fluid, Tissue Homogenate, and Other Body Fluids** *For Laboratory Research Use Only. Not Intended for Diagnostic Procedures.* This ELISA kit is designed for the quantitative determination of ENA-78 levels in various biological samples. The assay is based on the enzyme-linked immunosorbent assay (ELISA) principle, where a colorimetric reaction is used to measure the concentration of ENA-78. The Stop Solution changes the reaction from blue to yellow, and the optical density (OD) is measured at 450 nm using a microplate reader. To ensure accurate results, a set of calibration standards is included in the kit. These standards are run alongside the samples to generate a standard curve, which allows for the calculation of ENA-78 concentrations in unknown samples. This method ensures reliable and reproducible quantification of ENA-78 in research settings. **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. Remove serum and analyze immediately or 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. - **Cell Culture Supernatants, Tissue Homogenates, and Other Biological Fluids:** Centrifuge to remove particulates. Analyze immediately or store at -20°C. Avoid freeze-thaw cycles. **Note:** Ensure proper centrifugation, and avoid hemolysis or granulation in samples. **Materials Required but Not Supplied:** 1. Incubator 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 (Stored at 2–8°C):** - Microtiter Strip Plate (12×8 or 12×4 strips) - Standards (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 A (6.0 mL or 3.0 mL) - Chromogen 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 per kit) **Standard Concentrations:** 200, 100, 50, 25, 12.5, 6.25 pg/mL **Note:** If sample values exceed the highest standard, dilute with Sample Diluent and repeat the test. **Precautions:** - Do not mix reagents from different kit lots. - Allow all reagents to reach room temperature (20–25°C) before use. - Do not use reagents beyond their expiration date. - Keep unused strips in sealed bags with desiccant. - Wear gloves when handling biological samples. - Dispose of waste properly by adding 1% sodium hypochlorite and allowing it to sit for 30 minutes. - Avoid contact with acids and bleach. - Chromogen A and B contain 20% acetone; keep away from heat or flame. **Reagent Preparation:** - **Wash Solution (1X):** Dilute 1 volume of 20X Wash Solution with 19 volumes of distilled or deionized water. Store at 2–8°C for up to one month. **Assay Procedure:** 1. Prepare all reagents before starting. Run standards and samples in duplicate. 2. Add 50 µL of standard or sample to appropriate wells. Blank well receives no addition. 3. Add 100 µL of HRP-conjugate reagent to all wells except blank. Cover with adhesive strip and incubate at 37°C for 60 minutes. 4. Wash the plate 4 times manually or using automated washer. 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. Gently mix if color change is uneven. 7. Measure OD at 450 nm. Plot average OD values against standard concentrations to generate a standard curve. **Interpretation:** - Calculate mean OD for each standard and sample. Subtract blank OD from all readings. - Determine sample concentration by comparing its OD to the standard curve. - Intra-assay and inter-assay CV% are less than 15%. - Assay range: 6.25–200 pg/mL - Sensitivity: <1.0 pg/mL - Cross-reactivity: No significant cross-reactivity observed. **Storage:** - 2–8°C for frequent use; 6 months at -20°C **Important Note:** This kit is intended for research purposes only and should not be used for diagnostic or therapeutic applications. Always follow good laboratory practices and handle all biological materials with caution.

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