**Human HAI-1 ELISA Kit – For the Quantitative In Vitro Determination of Human Hepatocyte Growth Factor Activator Inhibitor Type 1 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 instruction manual carefully. This ELISA kit is designed for research purposes only and should not be used in clinical diagnostics.
The HAI-1 ELISA Kit utilizes an enzyme-linked immunosorbent assay (ELISA) to quantitatively measure the concentration of Human Hepatocyte Growth Factor Activator Inhibitor Type 1 (HAI-1) in biological samples. The detection process involves a colorimetric reaction, where the addition of a stop solution changes the color from blue to yellow. The optical density (OD) is then measured at 450 nm using a microplate reader. A standard curve is generated using a set of calibration standards, allowing for accurate quantification of HAI-1 levels in test samples 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. Centrifuge at 2000×g for 20 minutes. Remove serum and analyze immediately or store at -20°C. Avoid repeated freeze-thaw cycles.
- **Plasma:** Collect using heparin as an anticoagulant. Centrifuge within 30 minutes at 2000×g for 30 minutes at 2–8°C. Store at -20°C. Avoid repeated freeze-thaw.
- **Cell Culture Supernatants, Tissue Homogenates, and Other Biological Fluids:** Centrifuge to remove particulates and analyze immediately or store at -20°C. Avoid freeze-thaw cycles.
**Note:** Ensure adequate centrifugation and avoid hemolysis or granules in the sample.
**Materials Required but Not Supplied:**
- 37°C incubator
- Microplate reader capable of measuring absorbance at 450 nm
- Precision pipettes, disposable tips, and absorbent paper
- 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 |
**Standard Concentration:** 240, 120, 60, 30, 15, 7.5 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. Standards, conjugates, and plates are matched for optimal performance.
2. Bring all reagents and materials to room temperature (20–25°C) before use. Do not thaw using water baths.
3. Do not use reagents beyond their expiration date.
4. Handle all blood-derived samples as potentially infectious. Wear gloves during the procedure.
5. Do not remove microtiter plates from storage bags until needed. Store unused strips with desiccant at 2–8°C.
6. Use fresh pipette tips for each step.
7. Avoid contact with strong acids and sodium hypochlorite.
8. Waste must be inactivated for at least 30 minutes before disposal.
9. Chromogen solutions contain 20% acetone; keep away from heat and 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 the assay. Run standards and samples in duplicate.
2. Add 50 µL of standard or sample to the appropriate wells. The 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 microtiter plate 4 times manually or automatically.
5. Add 50 µL of Chromogen A and 50 µL of Chromogen B to each well. Incubate for 15 minutes at 37°C, protected from light.
6. Add 50 µL of Stop Solution to each well. Gently tap if color change is uneven.
7. Read OD at 450 nm. Plot average OD values against standard concentrations to generate a standard curve.
8. Calculate mean OD for each standard and sample. Subtract blank OD from all readings.
9. Locate the OD value on the Y-axis and draw a line to intersect the standard curve. Read the corresponding HAI-1 concentration.
**Performance Characteristics:**
- **Intra-Assay Range:** 7.5 ng/mL – 240 ng/mL
- **Sensitivity:** <1.0 ng/mL
- **Cross-Reactivity:** No significant cross-reactivity observed
- **Storage:** 2–8°C (frequent use); -20°C (long-term)
**Important Note:** Each user should generate their own standard curve due to possible variations in technique or environmental conditions. Always follow good laboratory practices when handling biological materials.
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