**Human LTF ELISA Kit – For the Quantitative In Vitro Determination of Human Lactoferrin in Serum, Plasma, Cerebrospinal Fluid, Tissue Homogenate, and Other Body Fluids**
**For Laboratory Research Use Only. Not for Diagnostic or Therapeutic Purposes.**
Before using this product, please read the entire package insert carefully. This kit is designed for research purposes only and should not be used in clinical diagnostic procedures. The test is based on the enzyme-linked immunosorbent assay (ELISA) principle, which allows for the quantitative measurement of lactoferrin (LTF) levels in biological samples.
The Stop Solution changes the color from blue to yellow, and the optical density (OD) is measured at 450 nm using a microplate reader. A set of calibration standards is included to generate a standard curve, which is essential for determining the concentration of LTF in unknown samples. By comparing the OD values of the samples with the standard curve, accurate quantification of LTF can be achieved.
**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 (20 minutes at ~2000×g). 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, 2–8°C. Store at -20°C. Avoid freeze-thaw cycles.
- **Cell Culture Supernatants, Tissue Homogenates, and Other Biological Fluids**: Centrifuge to remove particulates and 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.
| Name | 96 Determinations | 48 Determinations |
|------|-------------------|-------------------|
| MicroELISA Stripplate | 12×8 strips | 12×4 strips |
| Standard (6 Vial) | 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: 8000, 4000, 2000, 1000, 500, 250 μg/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 use water baths for thawing.
3. Do not use reagents past their expiration date.
4. Use only deionized or distilled water for dilutions.
5. Keep unused microtiter plate strips in the sealed bag with desiccant.
6. Use fresh pipette tips for each transfer to prevent cross-contamination.
7. Wear disposable gloves during the procedure. All biological samples should be handled with caution.
8. Dispose of all samples and waste according to local biohazard regulations.
9. Liquid waste must be inactivated by adding sodium hypochlorite (final concentration 1.0%) and allowing it to stand for 30 minutes.
10. Substrate solutions are sensitive. Discard if discolored.
11. Chromogen B contains 20% acetone—keep away from heat or flame.
12. Allow all reagents to equilibrate to room temperature before use.
**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 starting.
2. Add 100 µl of standards, samples, and blank to the microtiter plate. Cover with adhesive strip and incubate for 60 minutes at 37°C.
3. Wash the plate 4 times manually or automatically.
- **Manual Washing**: Aspirate contents, fill with 1X Wash Solution, aspirate again. Repeat 4 times.
- **Automated Washing**: Aspirate and wash 4 times with 350 µl/well.
4. After washing, 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.
5. Add 50 µl of Stop Solution to each well. The color will turn yellow. Gently tap if uneven.
6. Measure OD at 450 nm within 15 minutes.
**Calculation of Results**
1. Plot the average OD values of the standards against their concentrations.
2. Subtract the blank OD from all readings.
3. Use graph paper or software to construct the standard curve.
4. Determine the sample concentration by interpolation.
5. Intra-assay and inter-assay CVs are <15%.
6. Assay range: 250–8000 µg/mL.
7. Sensitivity: <100 µg/mL.
8. Cross-reactivity: No significant interference observed.
9. Storage: 2–8°C (frequent use); -20°C for long-term storage (up to 6 months).
**Important Notes**
- Always follow good laboratory practices.
- Handle all biological materials with care.
- Proper disposal of waste is critical.
This kit provides a reliable method for quantifying human lactoferrin in various biological matrices. It is ideal for research applications requiring accurate and reproducible results.
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