**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 carefully before using the Human Lp-PL-A2 ELISA Kit. The kit is designed for research purposes only and should not be used for diagnostic or therapeutic applications.
**INTENDED USE AND TEST PRINCIPLE**
The Human Lp-PL-A2 ELISA Kit is intended for laboratory research use only and is not suitable for diagnostic or clinical procedures. The test is based on the principle of a competitive enzyme-linked immunosorbent assay (ELISA). The color change from blue to yellow occurs after the addition of the Stop Solution, 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, the kit includes a set of calibration standards. These standards are run alongside the samples, allowing the operator to generate a standard curve of optical density (OD) versus Lp-PL-A2 concentration. The Lp-PL-A2 levels in the unknown samples are then calculated by comparing their OD values to the standard 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. 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 and assay immediately or store at -20°C. Avoid repeated freeze-thaw cycles.
- **Note**: Ensure proper centrifugation and avoid hemolysis or granulation in the samples.
**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
**REAGENTS PROVIDED**
All reagents are stored at 2–8°C. Refer to the expiration date on the label.
| 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 substitute reagents between different kit lots.
2. Allow all reagents to reach room temperature (20–25°C) before use.
3. Do not use reagents beyond their expiration date.
4. Use only deionized or distilled water for dilutions.
5. Keep microtiter plates in sealed bags until needed. Unused strips should be stored at 2–8°C with desiccant.
6. Use fresh pipette tips for each transfer to prevent contamination.
7. All disposable items must be handled as potentially hazardous.
8. Follow good laboratory practices when handling blood-derived materials.
9. Dispose of all samples properly to inactivate viruses.
10. Liquid waste should be treated with sodium hypochlorite (final concentration 1.0%) for 30 minutes before disposal.
11. Substrate solution may be contaminated; discard if it appears bluish.
12. Substrate B contains 20% acetone; keep away from heat and flame.
13. 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. Stable for 1 month at 2–8°C.
**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 the blank. Cover with adhesive strip and incubate for 60 minutes at 37°C.
4. Wash the plate 4 times.
- **Manual Washing**: Aspirate, fill with Wash Solution, aspirate again. Repeat four times.
- **Automated Washing**: Aspirate, wash four times with 350 μL/well.
5. After final wash, 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.
6. Add 50 μl of Stop Solution to each well. Color changes from blue to yellow. If green or uneven, gently tap the plate.
7. Measure OD at 450 nm within 15 minutes.
**CALCULATION OF RESULTS**
1. Plot the average OD (450 nm) of each standard against its concentration to generate a standard curve.
2. Subtract blank OD from all measurements.
3. Use graph paper or software to determine sample concentrations.
4. Variations in technique, time, or temperature may affect results. Each user should create their own standard curve.
5. Intra-assay and Inter-assay CV (%) < 15%.
6. Assay range: 1.56 ng/ml – 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 for up to 6 months.
**FOR RESEARCH USE ONLY; NOT FOR THERAPEUTIC OR DIAGNOSTIC APPLICATIONS!**
**PLEASE READ THROUGH ENTIRE PROCEDURE BEFORE BEGINNING!**
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