**Human LTF ELISA Kit – For the quantitative in vitro determination of Human Lactoferrin concentrations in serum, plasma, cerebrospinal fluid, tissue homogenate, and other body fluids. Intended for laboratory research use only. NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES.**
Before using this product, please read the entire package insert carefully.
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### **INTENDED USE AND TEST PRINCIPLE**
This LTF ELISA Kit is specifically designed for research purposes in a laboratory setting and should not be used for diagnostic or therapeutic applications. The assay is based on the principle of competitive immunoassay, where the concentration of lactoferrin (LTF) in the sample is determined by comparing its optical density (OD) to a standard curve generated from known concentrations of LTF.
The reaction is terminated with a Stop Solution, which changes the color from blue to yellow. The intensity of the yellow color is measured at 450 nm using a spectrophotometer. A set of calibration standards is included in the kit to create a reference curve, allowing for accurate quantification of LTF levels in unknown samples.
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### **SAMPLE COLLECTION AND STORAGE**
- **Serum:** Use a serum separator tube. Allow the blood 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 of collection at 2000×g for 30 minutes at 2–8°C. Store at -20°C. Avoid freeze-thaw cycles.
- **Cell culture supernatants, tissue homogenates, and other biological fluids:** Remove particulates by centrifugation. Assay immediately or aliquot and store at -20°C. Ensure no hemolysis or granules are present in the samples.
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### **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
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### **REAGENTS PROVIDED**
| Reagent Name | 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 |
*Note: Standard concentrations are 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 substitute reagents from different kits. All components are matched for optimal performance.
2. Allow all reagents and materials to reach room temperature (20–25°C) before use. Avoid using water baths for thawing.
3. Do not use any reagents beyond their expiration date.
4. Only use deionized or distilled water for reagent dilution.
5. Keep microtiter plates in their sealed bags until needed. Store unused strips at 2–8°C with desiccant.
6. Use fresh pipette tips for each transfer to prevent contamination.
7. Wear disposable gloves during the procedure. Treat all biological samples as potentially infectious.
8. Dispose of all samples and waste according to local regulations. Inactivate viral contaminants by adding 1% sodium hypochlorite to liquid waste and letting it stand for 30 minutes.
9. Substrate solution may be easily contaminated. Discard if it appears bluish before use.
10. Chromogen B contains 20% acetone; keep away from heat or flame.
---
### **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. Add samples and standards to the microplate. Cover with adhesive strips and incubate for 60 minutes at 37°C.
2. Wash the plate 4 times manually or automatically using 1X Wash Solution.
3. 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.
4. Add 50 µl of Stop Solution to each well. The color should turn yellow. If green or uneven, gently mix.
5. Measure OD at 450 nm within 15 minutes using a microplate reader.
---
### **CALCULATION OF RESULTS**
1. Plot the average OD values of the standards against their concentrations to generate a standard curve.
2. Subtract the blank OD from all readings before calculating results.
3. Use graph paper or software to determine the sample concentration.
4. Intra-assay and inter-assay CVs are less than 15%.
5. Assay range: 250–8000 μg/mL.
6. Sensitivity: <100 μg/mL.
7. Cross-reactivity: No significant cross-reactivity observed.
8. Storage: 2–8°C (frequent use); -20°C for long-term storage (up to 6 months).
---
**Always follow proper safety protocols and dispose of all waste according to local regulations. This kit is intended for research use only and must not be used in clinical settings.**
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