**Human LPA ELISA Kit – For the Quantitative In Vitro Determination of Human Lysophosphatidic Acid (LPA) Concentrations in Serum, Plasma, Cerebrospinal Fluid, Tissue Homogenate, and Other Body Fluids**
*For Laboratory Research Use Only. Not for 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 should not be used in clinical diagnostic procedures. The assay is based on the enzyme-linked immunosorbent assay (ELISA) principle, which allows for the quantitative measurement of LPA levels in biological samples.
The test works by detecting the concentration of LPA through a colorimetric reaction. A standard curve is generated using known concentrations of LPA, and the optical density (OD) values of unknown samples are compared to this curve to determine their LPA levels. The Stop Solution changes the color from blue to yellow, and the intensity of the color correlates with the LPA concentration.
**Sample Collection and Storage:**
- **Serum**: Collect using a serum separator tube. Allow clotting for 2 hours at room temperature or overnight at 4°C before centrifugation at 2000×g for 20 minutes. Assay immediately or 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.
- **Cell Culture Supernatants, Tissue Homogenates, and Other Biological Fluids**: Centrifuge to remove particulates and assay immediately or store at -20°C. Ensure no hemolysis or contamination occurs.
**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 (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 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:** 3200, 1600, 800, 400, 200, 100 pmol/mL. If sample values exceed the highest standard, dilute with Sample Diluent and repeat the assay.
**Precautions:**
- Do not mix reagents from different kits. All components are matched for optimal performance.
- Allow all reagents to reach room temperature (20–25°C) before use. Do not use water baths for thawing.
- Do not use reagents beyond their expiration date.
- Use only deionized or distilled water for dilution.
- Keep unused microtiter plate strips in sealed bags with desiccant.
- Always use fresh pipette tips to avoid cross-contamination.
- Treat all biological samples as potentially infectious. Follow good laboratory practices.
- Dispose of waste properly after inactivation for at least 30 minutes.
- Substrate solutions may be easily contaminated; keep away from heat or flame.
**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. Add 50 µl of standard or sample to the appropriate wells. Cover with an adhesive strip and incubate for 60 minutes at 37°C.
2. Wash the microtiter plate 4 times using either manual or automated washing methods.
3. 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.
4. Add 50 µl of Stop Solution to each well. Read OD at 450 nm using a microplate reader.
**Data Interpretation:**
- Plot average OD values of standards vs. LPA concentrations.
- Subtract blank OD from all readings before plotting.
- Calculate LPA concentration by comparing sample OD to the standard curve.
- Intra-assay and inter-assay CV% are less than 15%.
- Sensitivity: <100 pmol/mL. No significant cross-reactivity observed.
**Storage:**
- Store at 2–8°C for frequent use. For long-term storage, keep at -20°C. Shelf life: 6 months.
**Note:** Always follow the instructions provided in the user manual for accurate results. This kit is intended for research purposes only.
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