Human MBL ELISA Kit

**Human MBL ELISA Kit – For the Quantitative In Vitro Determination of Human Mannan-Binding Lectin (MBL) 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 product, please read the entire package insert carefully. This ELISA (Enzyme-Linked Immunosorbent Assay) is intended for research purposes only and should not be used in diagnostic or clinical settings. The MBL ELISA Kit allows for the accurate quantification of MBL levels in biological samples. The assay is based on a competitive immunoassay format, where the color change from blue to yellow is detected by measuring optical density (OD) at 450 nm. A standard curve is generated using a series of calibration standards with known MBL concentrations. By comparing the OD values of the test samples to this curve, the MBL concentration in the sample can be accurately determined. **Sample Collection and Storage:** - **Serum:** Collect using a serum separator tube. Allow clotting for 2 hours at room temperature or overnight at 4°C. Centrifuge at 2000×g for 20 minutes. Assay 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 for 30 minutes at 2–8°C. Store at -20°C. Avoid repeated 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 granulation occurs. **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):** - Microtiter Strip Plate (12×8 strips / 12×4 strips) - Standards (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 unit) **Notes:** - Standard concentrations: 400, 200, 100, 50, 25, 12.5 ng/mL - If sample values exceed the highest standard, dilute with Sample Diluent and repeat the assay - Always use reagents provided by the manufacturer - Allow all reagents and samples to reach room temperature (20–25°C) before use - Do not use water baths to thaw samples or reagents - Do not use expired kit components - Use only deionized or distilled water for reagent dilution - Keep microtiter plates in their sealed bag until needed - Store unused strips at 2–8°C with desiccant - Use fresh pipette tips for each transfer to avoid contamination - Wear gloves during the procedure; treat all blood-derived products as potentially infectious - Dispose of all samples according to local biohazard regulations - Waste must be treated with sodium hypochlorite (final concentration 1.0%) for at least 30 minutes before disposal - Substrate solutions are sensitive to contamination. Avoid exposure to heat or flame - Allow all reagents to reach 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 1 month. **Assay Procedure:** 1. Prepare all reagents before beginning the assay. It is recommended to run standards and samples in duplicate. 2. Add 20 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 the blank. Cover with adhesive strip and incubate for 60 minutes at 37°C. 4. Wash the plate 4 times: - Manual: Aspirate, fill with 1X Wash Solution, aspirate again. Repeat 4 times. Dry by blotting. - Automated: Use 1X Wash Buffer. Adjust brush to aspirate as much liquid as possible. Fill 350 µl/well/wash. 5. Add 50 µl of Chromogen A and 50 µl of Chromogen B to each well. Mix gently and incubate for 15 minutes at 37°C, away from light. 6. Add 50 µl of Stop Solution to each well. The color should turn from blue to yellow. If uneven, gently tap the plate. 7. Measure OD at 450 nm within 15 minutes using a microplate reader. **Calculation of Results:** 1. Plot average OD (450 nm) of each standard against concentration to generate a standard curve. 2. Calculate mean OD for each standard and sample. Subtract blank OD before interpretation. 3. Use graph paper or software to determine MBL concentration. 4. Result variability may occur due to differences in technique, incubation time, or kit age. Each user should prepare their own standard curve. 5. Intra-assay and inter-assay CV% are <15%. 6. Assay range: 12.5 ng/mL – 400 ng/mL. 7. Sensitivity: <10 ng/mL. 8. No significant cross-reactivity observed. 9. Storage: 2–8°C (for frequent use); 6 months at -20°C. **Important Note:** This product is for research use only. Proper training and safety precautions must be followed when handling biological materials. Always refer to the latest version of the manual for complete instructions.

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