**Human Lp-PL-A2 ELISA Kit – For the Quantitative In Vitro Determination of Human Lipoprotein-Associated Phospholipase A2 Concentrations in Serum, Plasma, Celiac Fluid, Tissue Homogenate, and Body Fluids**
**FOR LABORATORY RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES.**
This package insert must be read thoroughly before use. This ELISA kit is designed for research purposes only and is not intended for diagnostic or therapeutic applications. The method is based on a competitive enzyme-linked immunosorbent assay (ELISA) principle.
The Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm using a spectrophotometer. To determine the concentration of Lp-PL-A2 in the sample, this kit includes a set of calibration standards. These standards are run alongside the samples to generate a standard curve, which is used to calculate the concentration of Lp-PL-A2 in the unknown samples by comparing their optical density (OD) values.
**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. Remove the serum and assay immediately, or aliquot and store at -20°C. Avoid repeated freeze-thaw cycles.
- **Plasma**: Collect plasma 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 freeze-thaw cycles.
- **Cell culture supernatants, tissue homogenates, and other biological fluids**: Centrifuge to remove particulates, then assay immediately or store at -20°C. Avoid repeated freeze-thaw cycles.
*Note: Ensure adequate centrifugation and avoid hemolysis or granulation in the samples.*
**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
**REAGENTS PROVIDED (STORED AT 2–8°C)**
| 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: 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.*
**PRECAUTIONS**
1. Do not mix reagents from different kit lots.
2. Allow all reagents and materials to reach room temperature (20–25°C) before use.
3. Do not use reagents beyond the 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 pipette tips for each transfer to prevent contamination.
7. Dispose of all waste properly, considering potential infectious risk.
8. All samples should be handled as potentially infectious. Follow good laboratory practices.
9. Inactivate viruses in liquid waste by adding 1% sodium hypochlorite and allowing it to stand for 30 minutes.
10. Substrate solutions should be checked for contamination before use.
11. Chromogen B contains 20% acetone; keep away from heat or flame.
12. Allow all reagents to reach room temperature before use.
**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.
**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: no addition.
3. Add 100 µl of HRP-conjugate reagent to all wells except blank. Cover with adhesive strip and incubate for 60 minutes at 37°C.
4. Wash plate 4 times manually or automatically.
- *Manual Washing:* Aspirate, fill with 1X Wash Solution, and repeat four times. Blot dry after final wash.
- *Automated Washing:* Aspirate and wash four times. Adjust brush to aspirate maximum liquid.
5. 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.
6. Add 50 µl of Stop Solution to each well. Color changes from blue to yellow. If uneven, gently tap the plate.
7. Read OD at 450 nm within 15 minutes using a microplate reader.
**CALCULATION OF RESULTS**
1. Plot average OD (450 nm) against standard concentrations to create a standard curve.
2. Subtract blank OD from all readings before interpreting results.
3. Locate the sample OD on the Y-axis and draw a horizontal line to intersect the standard curve. Read the corresponding concentration on the X-axis.
4. Each user should generate their own standard curve due to possible variations in technique.
5. Intra-assay and inter-assay CV% < 15%.
6. Assay range: 1.56–50 ng/ml.
7. Sensitivity: <1.0 ng/ml.
8. Cross-reactivity: No significant cross-reactivity observed.
9. Storage: 2–8°C (frequent use); -20°C (long-term).
**FOR RESEARCH USE ONLY; NOT FOR THERAPEUTIC OR DIAGNOSTIC APPLICATIONS!**
**PLEASE READ THROUGH THE ENTIRE PROCEDURE BEFORE BEGINNING!**
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