**Human LPS ELISA Kit – For the Quantitative In Vitro Determination of Human Lipopolysaccharides (LPS) Concentrations in Serum, Plasma, Cerebrospinal Fluid, Tissue Homogenate, and Other Body Fluids**
**For Laboratory Research Use Only. NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES.**
Before using this product, please read the entire package insert carefully. This ELISA kit is designed for research purposes only and is not intended for diagnostic or clinical use. The test principle is based on a sandwich immunoassay format. The color change from blue to yellow occurs after adding the Stop Solution, and the intensity of the color correlates with the concentration of LPS in the sample. Calibration standards are run alongside the samples to generate a standard curve, allowing accurate quantification of LPS levels by comparing the optical density (OD) of the samples to the curve.
**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 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 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 debris remains.
**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
- Standard (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 A: 6.0 ml / 3.0 ml
- Chromogen 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**
1. Standard concentrations: 800, 400, 200, 100, 50, 25 ng/mL.
2. If sample values exceed the highest standard, dilute with Sample Diluent and repeat the assay.
3. All reagents should reach room temperature (20–25°C) before use. Do not thaw using water baths.
4. Do not use reagents past their expiration date.
5. Use only deionized or distilled water for reagent dilutions.
6. Keep microtiter plate in its sealed bag until use. Unused strips must be stored with desiccant at 2–8°C.
7. Use fresh pipette tips for each transfer to avoid cross-contamination.
8. Wear disposable gloves throughout the procedure. All biological materials should be treated as potentially infectious.
9. Dispose of all samples following proper biohazard protocols.
10. Liquid waste: Add sodium hypochlorite to a final concentration of 1.0% and allow to stand for at least 30 minutes before disposal.
11. Substrate solution may become contaminated. Discard if it appears bluish.
12. Chromogen B contains 20% acetone—keep away from heat and flame.
13. Allow all reagents to reach room temperature before starting the assay.
**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.
2. Add samples and standards to the microtiter plate. Cover with adhesive strips and incubate for 60 minutes at 37°C.
3. Wash the plate four times manually or automatically.
- **Manual Washing**: Aspirate and fill each well with 1X Wash Solution, repeating four times. Blot dry.
- **Automated Washing**: Aspirate and wash four times, ensuring 350 μL per well.
4. Add 50 μL of Chromogen A and 50 μL of Chromogen B to each well. Incubate for 15 minutes at 37°C, protecting from light.
5. Add 50 μL of Stop Solution to each well. The color will change from blue to yellow. If uneven, gently tap the plate.
6. Measure OD at 450 nm within 15 minutes.
**Calculation of Results**
1. Plot the average OD values of standards against their concentrations to create a standard curve.
2. Subtract blank OD from all readings.
3. Use graph paper or software to determine LPS concentration in unknown samples.
4. Intra-assay and inter-assay CV% are less than 15%.
5. Assay range: 25 ng/mL to 800 ng/mL.
6. Sensitivity: <10 ng/mL.
7. Cross-reactivity: No significant interference observed.
8. Storage: 2–8°C (frequent use); 6 months at -20°C.
**Important Safety Information**
Always follow good laboratory practices when handling biological samples. Treat all reagents and samples as potentially hazardous. Follow local regulations for waste disposal.
This kit is ideal for researchers studying inflammation, immune responses, and bacterial infections. It provides a reliable and sensitive method for detecting LPS levels in various biological matrices. Make sure to follow all instructions carefully to ensure accurate and reproducible results.
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