**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 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 diagnostic settings.
**Intended Use and Test Principle**
This MBL ELISA Kit is intended for the quantitative determination of human mannan-binding lectin (MBL) in various biological samples. The test works by creating a standard curve using known concentrations of MBL. The optical density (OD) values obtained from the sample are then compared to this curve to calculate the MBL concentration.
The Stop Solution changes the color from blue to yellow, and the intensity of the color is directly proportional to the MBL concentration. A microplate reader is used to measure absorbance at 450 nm.
**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. Store at -20°C after aliquoting. 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 Fluids**: Centrifuge to remove particulates. Assay immediately or store at -20°C. Ensure no hemolysis or granules are 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**
All reagents should be stored at 2–8°C. Check expiration date on the label.
| Reagent | 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**: 400, 200, 100, 50, 25, 12.5 ng/mL. If sample values exceed the highest standard, dilute with Sample Diluent and repeat the assay.
**General Instructions**
1. Use only manufacturer-supplied reagents.
2. Allow all reagents and samples to reach room temperature (20–25°C) before use. Do not thaw using water baths.
3. Do not use reagents beyond their expiration date.
4. Use only deionized or distilled water for dilutions.
5. Keep unused strip plates in sealed bags with desiccant at 2–8°C.
6. Use fresh pipette tips for each transfer to prevent cross-contamination.
7. Wear gloves during the procedure. All biological materials should be considered potentially infectious.
8. Dispose of all waste properly. Add sodium hypochlorite to a final concentration of 1.0% and allow it to stand for 30 minutes before disposal.
9. Substrate solutions may be easily contaminated. Handle with care.
10. Avoid heat or flame near Chromogen B, which contains 20% acetone.
**Reagent Preparation**
- **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 20 µl of standard or sample to each well. 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 plate 4 times manually or via automated system. After final wash, blot dry.
5. Add 50 µl of Chromogen A and 50 µl of Chromogen B to each well. Incubate at 37°C for 15 minutes, protecting from light.
6. Add 50 µl of Stop Solution. Color should change from blue to yellow. Gently mix if necessary.
7. Read OD at 450 nm within 15 minutes.
**Calculation of Results**
1. Plot average OD (450 nm) against standard concentrations to generate a standard curve.
2. Subtract blank OD from all readings before interpreting results.
3. Calculate mean OD for each standard and sample.
4. Construct standard curve using graph paper or software. Determine unknown concentration by interpolation.
5. Intra-assay and inter-assay CV (%) are less than 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 (frequent use); -20°C (long-term).
10. Always follow safety protocols and proper waste disposal procedures.
**Note**: This product is intended for research use only and must not be used in diagnostic or therapeutic applications.
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