**Human LPS ELISA Kit – For the Quantitative In Vitro Determination of Human Lipopolysaccharide (LPS) 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 kit is designed for research purposes only and should not be used in clinical diagnostic settings.
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### **Intended Use and Test Principle**
This LPS ELISA Kit is intended for laboratory research use only and is not suitable for diagnostic or therapeutic applications. The method is based on the enzyme-linked immunosorbent assay (ELISA) principle. The color change from blue to yellow upon addition of the Stop Solution indicates the endpoint of the reaction. A standard curve is generated using known concentrations of LPS standards, which allows the quantification of unknown samples by comparing their optical density (OD) values at 450 nm.
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### **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 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 of collection at 2000×g for 30 minutes at 2–8°C. Store at -20°C. Avoid repeated freezing.
- **Cell Culture Supernatants, Tissue Homogenates, and Other Biological Fluids**: Remove particulates by centrifugation. Assay immediately or store at -20°C. Ensure no hemolysis or granules are present.
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### **Materials Required but 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
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### **Reagents Provided**
All reagents should be stored at 2–8°C. Check the expiration date on the label.
| 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 |
**Notes:**
- Standard concentrations: 800, 400, 200, 100, 50, 25 ng/mL.
- If sample values exceed the highest standard, dilute with Sample Diluent and repeat the assay.
- All reagents should reach room temperature (20–25°C) before use. Do not thaw using water baths.
- Do not use reagents past their expiration date.
- Use only deionized or distilled water for dilution.
- Keep microtiter plate in sealed bag until needed. Unused strips should be stored at 2–8°C with desiccant.
- Always use fresh pipette tips to prevent cross-contamination.
- Wear disposable gloves throughout the procedure.
- Treat all biological samples as potentially infectious.
- Dispose of all waste following proper inactivation protocols.
- Substrate B contains 20% acetone; keep away from heat and flame.
- Ensure all reagents are at room temperature before use.
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### **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.
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### **Assay Procedure**
1. Prepare all reagents before starting.
2. Add samples and standards 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: Aspirate, fill with Wash Solution, aspirate again. Repeat 4 times. Dry by blotting.
- Automated: Use 350 μL/well for each wash. Dry after final wash.
4. 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, protecting from light.
5. Add 50 μL of Stop Solution to each well. The color should turn yellow. If green or uneven, gently tap the plate.
6. Measure OD at 450 nm within 15 minutes.
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### **Calculation of Results**
1. Plot the average OD values of the standards on a graph (Y-axis vs. X-axis).
2. Subtract blank OD values from all readings.
3. Determine the LPS concentration of each sample by locating its OD on the Y-axis and finding the corresponding value on the X-axis.
4. Each user should generate their own standard curve.
5. Intra-assay and inter-assay CV% are <15%.
6. Assay range: 25 ng/mL – 800 ng/mL.
7. Sensitivity: <10 ng/mL.
8. Cross-reactivity: No significant interference observed.
9. Storage: 2–8°C (frequent use), 6 months at -20°C.
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### **Safety and Disposal**
- All samples must be treated as potentially infectious.
- Liquid waste: Add 1.0% sodium hypochlorite and let stand for 30 minutes before disposal.
- Discard any contaminated or expired reagents properly.
**Note:** This document is for reference only. Always follow the manufacturer’s instructions for accurate results.
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