**Human Lp-PL-A2 ELISA Kit – For the Quantitative In Vitro Determination of Human Lipoprotein-Associated Phospholipase A2 Concentrations in Serum, Plasma, Cerebrospinal Fluid, Tissue Homogenate, and Other Body Fluids**
**For Laboratory Research Use Only. Not for Diagnostic Procedures.**
Before using this product, please read the entire insert carefully. This ELISA kit is designed for research purposes only and should not be used for diagnostic or therapeutic applications. The assay is based on a colorimetric reaction, where the color changes from blue to yellow, and the intensity is measured at 450 nm using a spectrophotometer.
The kit includes a set of calibration standards that are run alongside the samples. These standards help create a standard curve by plotting optical density (OD) values against known concentrations of Lp-PL-A2. The concentration of Lp-PL-A2 in unknown samples is then determined by comparing their OD values to this standard curve.
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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 freeze-thaw cycles.
- **Cell Culture Supernatants, Tissue Homogenates, and Other Biological Fluids:** Centrifuge to remove particulates. Assay immediately or store at 2–8°C within 30 minutes. Avoid repeated freezing and thawing.
- **Note:** Ensure adequate centrifugation and avoid hemolysis or contamination with cellular debris.
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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 | 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 |
**Standard Concentrations:** 50, 25, 12.5, 6.25, 3.12, 1.56 ng/ml
**Note:** 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 kit lots. All components are calibrated for optimal performance.
2. Allow all reagents and samples to reach room temperature (20–25°C) before use. Do not use water baths to thaw.
3. Do not use any reagents beyond their expiration date.
4. Use only deionized or distilled water for dilution.
5. Keep microtiter plates in their sealed bags until ready to use. Store unused strips at 2–8°C with desiccant.
6. Use fresh disposable pipette tips for each step.
7. Handle serum and plasma as potentially infectious materials. Wear gloves during the procedure.
8. Waste must be inactivated for at least 30 minutes before disposal. Add 1% sodium hypochlorite to liquid waste.
9. Substrate solution may be contaminated; if it appears bluish, do not use.
10. Chromogen B contains 20% acetone—keep away from heat and flame.
11. Always allow reagents to warm to room temperature before starting the assay.
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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 beginning. 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 the blank. Cover with adhesive strip and incubate for 60 minutes at 37°C.
4. Wash the plate 4 times. Manual washing involves filling and aspirating each well. Automated washing requires 350 µl/well per wash.
5. After final wash, 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, protected from light.
6. Add 50 µl of Stop Solution to each well. If color is uneven, gently tap the plate.
7. Measure OD at 450 nm. Plot average OD values of standards vs. concentrations to generate a standard curve.
8. Calculate mean OD for each sample and subtract blank value before interpretation.
9. Locate the OD value on the Y-axis and draw a line to intersect the standard curve. Read the corresponding concentration.
10. Note that variations in technique, timing, or kit age may affect results. Each user should generate their own standard curve.
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**Performance Characteristics:**
- **Intra-assay CV (%) and Inter-assay CV (%):** <15%
- **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 (for frequent use); 6 months at -20°C
**Important:** Always follow the manufacturer’s instructions and safety guidelines. This product is intended for research use only and should not be used in clinical settings.
RYEOGLOBAL Co,.LTD , https://www.ryeolab.com