**Human vWF ELISA Kit – For the Quantitative In Vitro Determination of Human von Willebrand Factor Concentrations in Serum, Plasma, Cerebrospinal Fluid, Tissue Homogenate, and Other Body Fluids** **For Laboratory Research Use Only. Not for Diagnostic or Therapeutic Procedures.** Before using this kit, please read the entire package insert carefully. This ELISA (Enzyme-Linked Immunosorbent Assay) is designed for research purposes only and should not be used in clinical diagnostics. ### **Intended Use and Test Principle** This vWF ELISA Kit is intended for laboratory research use only and is not suitable for diagnostic or therapeutic applications. The test principle involves a competitive immunoassay where the concentration of vWF in the sample is determined by comparing its optical density (OD) to a standard curve generated from known concentrations of vWF. A stop solution is added to terminate the reaction, changing the color from blue to yellow. The intensity of the color is proportional to the vWF concentration. ### **Sample Collection and Storage** - **Serum**: Use serum separator tubes. Allow samples to clot for 2 hours at room temperature or overnight at 4°C before centrifugation at 2000×g for 20 minutes. Assay immediately or aliquot and 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. Avoid freeze-thaw cycles. - **Cell Culture Supernatants, Tissue Homogenates, 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 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 the expiration date on the label. - MicroELISA Strip Plate: 12×8 strips (96 determinations), 12×4 strips (48 determinations) - Standard (6 vials): 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 units - User Manual: 1 copy - Sealed Bags: 1 per kit ### **Important Notes** - Standard concentrations: 400, 200, 100, 50, 25, 12.5 U/L. - If sample values exceed the highest standard, dilute with Sample Diluent and repeat the test. - Do not thaw reagents in a water bath. - Do not use components beyond their expiration date. - Use only deionized or distilled water for reagent dilutions. - Keep microtiter plates in sealed bags until use. Store unused strips at 2–8°C with desiccant. - Use fresh pipette tips for each transfer to avoid cross-contamination. - Wear gloves during the procedure. All biological materials should be considered potentially infectious. - Dispose of all samples properly after testing. - Liquid waste must be treated with 1% sodium hypochlorite for at least 30 minutes before disposal. - Substrate solutions are sensitive; avoid exposure to heat or flame. - Bring all reagents to room temperature (20–25°C) before use. ### **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 1 month. ### **Assay Procedure** 1. Prepare all reagents before starting the assay. 2. Add 50 µL of standard or sample to appropriate wells (excluding blank). Cover with adhesive strip and incubate for 60 minutes at 37°C. 3. Wash the microtiter plate 4 times. - **Manual Washing**: Aspirate contents, fill with 1X Wash Solution, aspirate again. Repeat 4 times. Dry by blotting. - **Automated Washing**: Follow manufacturer instructions. Adjust brush to remove maximum liquid and set fill volume to 350 µL/well. 4. 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. 5. Add 50 µL of Stop Solution to each well. The color changes from blue to yellow. If uneven, gently tap the plate. 6. Measure OD at 450 nm within 15 minutes using a microplate reader. ### **Calculation of Results** - Plot average OD (450 nm) of standards against concentrations. - Subtract blank OD from all measurements. - Locate sample OD on Y-axis and find corresponding X-axis value on the standard curve. - Intra-assay and inter-assay CV (%) < 15%. - Assay range: 12.5 – 400 U/L. - Sensitivity: <10 U/L. - Cross-reactivity: No significant interference. - Storage: 2–8°C (frequent use); 6 months at -20°C. **Always follow good laboratory practices and handle all biological materials with care.**

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