**Human MFG-E8 ELISA Kit – For the Quantitative In Vitro Determination of Human Milk Fat Globule EGF Factor 8 Concentrations in Serum, Plasma, Cerebrospinal Fluid, Tissue Homogenate, and Other Body Fluids** *For Laboratory Research Use Only. Not for Diagnostic or Therapeutic Procedures.* This ELISA kit is designed to quantitatively measure Human MFG-E8 levels in various biological samples. The assay utilizes a colorimetric method where the reaction changes from blue to yellow upon addition of the Stop Solution, and the optical density (OD) is measured at 450 nm using a spectrophotometer. The kit includes a set of calibration standards that are run alongside the samples to generate a standard curve. This allows for accurate determination of MFG-E8 concentrations by comparing sample OD values against 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 centrifuging at 2000 ×g for 20 minutes. Remove serum and analyze 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). Avoid freezing and thawing. - **Cell Culture Supernatants, Tissue Homogenates, and Other Biological Fluids**: Centrifuge to remove particulates, then analyze immediately or store at -20°C. Ensure no hemolysis or debris is present. --- ### **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** | Reagent | 96 Determinations | 48 Determinations | |--------|------------------|-------------------| | MicroELISA Stripplate | 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 | *Note: Standard concentrations are 240, 120, 60, 30, 15, and 7.5 pg/mL. If sample values exceed the highest standard, dilute with Sample Diluent and repeat the assay.* --- ### **Precautions** 1. Do not mix reagents from different kit lots. All components are calibrated for optimal performance. 2. Allow all reagents to reach room temperature (20–25°C) before use. Do not thaw on water baths. 3. Do not use reagents beyond their expiration date. 4. Always use deionized or distilled water for dilutions. 5. Keep unused microtiter plate strips in their sealed pouch with desiccant at 2–8°C. 6. Use fresh pipette tips for each transfer to prevent cross-contamination. 7. Handle disposable plasma samples as potentially hazardous. Wear gloves during the procedure. 8. Dispose of all waste properly after inactivation (allow standing for 30 minutes). 9. Substrate solutions must be used before they turn bluish. 10. Chromogen B contains 20% acetone—keep away from heat or flame. 11. Ensure all reagents are at room temperature before starting the assay. --- ### **Reagent Preparation and Storage** - **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 before starting. Run standards and samples in duplicate. 2. Add 50 μL of standard or sample to the appropriate wells. Leave blank well empty. 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 microtiter plate 4 times manually or automatically. Ensure thorough washing. 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. The color should change from blue to yellow. If green or uneven, gently tap the plate. 7. Read OD at 450 nm. Generate a standard curve by plotting average OD values against standard concentrations. --- ### **Interpretation and Performance** - Plot the average OD (450 nm) on the Y-axis versus concentration on the X-axis. - Subtract blank OD from all measurements before calculating results. - Each user should generate their own standard curve due to potential variations in technique. - Intra-assay CV <15%, Inter-assay CV <15%. - Assay range: 7.5 pg/mL – 240 pg/mL. - Sensitivity: <1.0 pg/mL. - Cross-reactivity: No significant interference observed. - Storage: 2–8°C (frequent use), or -20°C for long-term storage (up to 6 months). --- **Note:** This product is intended solely for research purposes. It is not approved for clinical diagnostic use. Always follow proper laboratory safety protocols.

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