Human ENA-78 ELISA Kit

**Human ENA-78 ELISA Kit – For the Quantitative In Vitro Determination of Human Epithelial Neutrophil Activating Peptide 78 in Serum, Plasma, Cerebrospinal Fluid, Tissue Homogenate, and Other Biological Fluids** *For Laboratory Research Use Only. Not for Diagnostic or Therapeutic Procedures.* This ELISA kit is designed for the quantitative determination of Human ENA-78 (also known as CXCL5) concentrations in various biological samples. The method is based on a sandwich immunoassay format using enzyme-linked immunosorbent assay (ELISA). The color change from blue to yellow is triggered by the Stop Solution, and the optical density (OD) is measured at 450 nm with a spectrophotometer. A standard curve is generated using a set of calibration standards, allowing for accurate quantification of ENA-78 levels in test samples. --- **Intended Use** This ENA-78 ELISA Kit is intended for research purposes only and should not be used in diagnostic or therapeutic applications. It provides a reliable and sensitive method for measuring ENA-78 in human serum, plasma, cerebrospinal fluid, tissue homogenates, and other body fluids. **Test Principle** The assay employs a two-step immunoenzymatic reaction. Standards and samples are incubated in microtiter wells coated with anti-ENA-78 antibodies. After washing, a horseradish peroxidase (HRP)-conjugated secondary antibody is added, followed by a chromogenic substrate. The reaction is stopped, and the absorbance is read at 450 nm. The concentration of ENA-78 in the sample is determined by comparing its OD value to the standard curve. --- **Sample Collection and Storage** - **Serum**: Use a serum separator tube. Allow samples to clot for 2 hours at room temperature or overnight at 4°C before centrifugation at 2000×g for 20 minutes. Remove the 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 of collection at 2000×g for 30 minutes at 2–8°C. Store at -20°C. Avoid repeated freeze-thaw. - **Cell Culture Supernatants, Tissue Homogenates, and Other Body Fluids**: Centrifuge to remove particulates. Analyze immediately or store at -20°C. Avoid freeze-thaw cycles. - **Note**: Ensure adequate centrifugation; avoid hemolysis or granulation in samples. --- **Materials Required (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 (Stored at 2–8°C)** | 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**: 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 assay. --- **Precautions** 1. Do not substitute reagents between different kit lots. 2. Allow all reagents and samples to reach room temperature (20–25°C) before use. 3. Do not use reagents beyond their expiration date. 4. Keep microtiter plates in sealed bags until needed. Store unused strips at 2–8°C with desiccant. 5. Wear gloves during handling. Treat all biological samples as potentially infectious. 6. Dispose of waste properly after inactivation with 1.0% sodium hypochlorite. 7. Avoid contact with acids and bleach. 8. Handle chromogen solutions with care due to acetone content. 9. Avoid exposure to heat or flame when working with chromogen solutions. --- **Reagent Preparation and Storage** - **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 for 60 minutes at 37°C. 4. Wash the plate 4 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, protected from light. 6. Add 50 μL of Stop Solution to each well. Gently mix if color is uneven. 7. Read OD at 450 nm. **Data Interpretation** - Plot average OD values (450 nm) against standard concentrations to generate a standard curve. - Subtract blank OD from all readings before calculating results. - Locate the sample OD on the Y-axis and draw a line to intersect the standard curve. Read the corresponding concentration. - Intra-assay and inter-assay CV% < 15%. - Assay range: 6.25–200 pg/mL. - Sensitivity: <1.0 pg/mL. - Cross-reactivity: No significant cross-reaction observed. **Storage** - 2–8°C for frequent use; 6 months at -20°C. **Note**: Always refer to the user manual for detailed instructions and safety information.

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