**Human Lp-PL-A2 ELISA Kit – For the Quantitative In Vitro Determination of Human Lipoprotein-Associated Phospholipase A2 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 kit, please read the entire instruction manual carefully. This ELISA kit is designed for research purposes only and is not intended for clinical diagnosis. The detection method relies on a colorimetric reaction, where the color changes from blue to yellow, and the optical density (OD) is measured at 450 nm using a spectrophotometer.
The kit includes a set of calibration standards that are used to create a standard curve. By comparing the OD values of the samples with the standard curve, the concentration of Lp-PL-A2 can be accurately determined.
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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. Centrifuge at 2000×g for 20 minutes. Remove serum and assay immediately or aliquot and store at -20°C. Avoid repeated freeze-thaw cycles.
- **Plasma**: Collect using heparin as an anticoagulant. Centrifuge at 2000×g for 30 minutes. Avoid freezing and thawing multiple times.
- **Cell Culture Supernatants, Tissue Homogenates, and Other Biological Fluids**: Centrifuge to remove particulates and assay immediately or store at 2–8°C within 30 minutes of collection. Avoid hemolysis and granules.
Note: Ensure proper centrifugation and avoid any contamination or hemolysis during sample preparation.
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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 (Stored at 2–8°C)**
| Reagent Name | 96 Determinations | 48 Determinations |
|-------------------------------|-------------------|-------------------|
| MicroELISA Stripplate | 12x8 strips | 12x4 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: 50, 25, 12.5, 6.25, 3.12, 1.56 ng/ml.*
*If sample values exceed the highest standard, dilute with Sample Diluent and repeat the assay.*
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### **Precautions**
1. Do not mix reagents from different kits. All components are matched for optimal performance.
2. Allow all reagents and samples 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 dilution.
5. Keep microtiter plates in sealed bags until needed. Store unused strips at 2–8°C with desiccant.
6. Use fresh disposable pipette tips.
7. Avoid contact with strong acids or sodium hypochlorite.
8. Handle serum and plasma as potentially infectious. Wear gloves during the procedure.
9. Waste must be inactivated for at least 30 minutes before disposal.
10. Liquid waste: Add 1.0% sodium hypochlorite.
11. Substrate B contains 20% acetone—keep away from heat or flame.
12. Allow all reagents to reach 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 one month.
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### **Assay Procedure**
1. Prepare all reagents before starting. Run standards and samples in duplicate.
2. Add 50 µl of standard or sample to appropriate wells. 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 the plate 4 times manually or automatically:
- Manual: Fill each well with 1X Wash Solution, aspirate, and repeat 4 times.
- Automatic: Aspirate and fill 350 µL/well. Ensure thorough washing.
5. After final wash, invert and blot dry.
6. 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.
7. Add 50 µl of Stop Solution to each well. If color is uneven, tap the plate gently.
8. Measure OD at 450 nm.
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### **Data Interpretation**
- Plot the average OD values of standards against their concentrations to generate a standard curve.
- Subtract blank OD from all readings.
- Locate the sample OD on the Y-axis and draw a horizontal line to intersect the curve. Read the corresponding concentration on the X-axis.
- Each user should prepare their own standard curve due to possible variations in technique or environmental factors.
- Intra-assay and inter-assay CV% < 15%.
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### **Additional Information**
- **Assay Range**: 1.56 ng/ml – 50 ng/ml
- **Sensitivity**: < 1.0 ng/ml
- **Cross-Reactivity**: No significant cross-reactivity with other proteins.
- **Storage**: 2–8°C (frequent use); 6 months at -20°C.
- **Shelf Life**: 12 months from date of manufacture when stored properly.
Always follow safety protocols and handle all materials with care. This product is intended for research use only and should not be used for diagnostic or clinical purposes.
RYEOGLOBAL Co,.LTD , https://www.ryeolab.com