**Human HAI-1 ELISA Kit – For the Quantitative In Vitro Determination of Human Hepatocyte Growth Factor Activator Inhibitor Type 1 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 is not intended for clinical diagnosis or treatment.
The **HAI-1 ELISA Kit** uses a colorimetric method to quantify HAI-1 levels in biological samples. The reaction involves binding of HAI-1 to specific antibodies immobilized on a microplate. After incubation with a horseradish peroxidase (HRP)-conjugated detection antibody, a chromogenic substrate is added, producing a blue color that turns yellow upon addition of the stop solution. The optical density (OD) at 450 nm is measured using a spectrophotometer, and the concentration of HAI-1 in the sample is determined by comparing its OD value to a standard curve generated from known concentrations of HAI-1.
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### **INTENDED USE AND TEST PRINCIPLE**
This HAI-1 ELISA Kit is specifically designed for **laboratory research use only** and should not be used for diagnostic or therapeutic purposes. The assay relies on a competitive immunoassay format, where the presence of HAI-1 in the sample competes with the standard for binding sites on the antibody-coated microplate. A standard curve is constructed using serial dilutions of HAI-1 standards, allowing for accurate quantification of unknown samples.
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### **SAMPLE COLLECTION AND STORAGE**
- **Serum**: Collect using a serum separator tube. Allow clotting for 2 hours at room temperature or overnight at 4°C. Centrifuge at 2000×g for 20 minutes. Remove serum and assay immediately or store at -20°C. Avoid repeated freeze-thaw cycles.
- **Plasma**: Use 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. Avoid repeated freezing and thawing.
**Note:** Ensure proper centrifugation to avoid hemolysis or contamination with cellular debris.
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### **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
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### **REAGENTS PROVIDED (Storage: 2–8°C)**
| Reagent Name | 96 Determinations | 48 Determinations |
|--------------|-------------------|-------------------|
| MicroELISA Strip Plate | 12 x 8 strips | 12 x 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:** 240, 120, 60, 30, 15, 7.5 ng/mL
**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 to reach room temperature (20–25°C) before use. Do not use water baths for thawing.
3. Do not use reagents past their expiration date.
4. Store unused strips in sealed bags with desiccant at 2–8°C.
5. Wear disposable gloves during the procedure to prevent exposure to potentially infectious materials.
6. Handle all biological samples as potentially hazardous.
7. Waste must be inactivated for at least 30 minutes before disposal.
8. Chromogen Solutions A and B contain 20% acetone. Keep away from heat and flame.
9. Always wash plates four times during the procedure, ensuring complete removal of unbound reagents.
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### **REAGENT PREPARATION AND STORAGE**
- **Wash Solution (1X):** Dilute 1 volume of 20X Wash Solution with 19 volumes of distilled or deionized water. Store at 2–8°C for up to one month.
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### **ASSAY PROCEDURE**
1. Prepare all reagents before starting the assay. Add standards and samples in duplicate to the microplate.
2. Add 50 µL of standard or sample to each well. Blank wells receive 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:** Fill each well with 1X Wash Solution, aspirate, and repeat four times.
- **Automated Washing:** Aspirate all wells and wash four times with 350 µL/well of 1X Wash Solution.
5. After washing, add 50 µL of Chromogen Solution A and 50 µL of Chromogen Solution B to each well. Incubate for 15 minutes at 37°C, protecting from light.
6. Add 50 µL of Stop Solution to each well. Mix gently if color is uneven.
7. Read OD at 450 nm. Generate a standard curve by plotting average OD values against standard concentrations.
8. Calculate the mean OD for each sample and subtract the blank value. Determine HAI-1 concentration by interpolating from the standard curve.
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### **SPECIFICATIONS**
- **Assay Range:** 7.5 ng/mL – 240 ng/mL
- **Sensitivity:** <1.0 ng/mL
- **Cross-Reactivity:** No significant cross-reactivity observed with other proteins
- **Storage:** 2–8°C (for frequent use); -20°C (for long-term storage)
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**Important Note:** Each user should generate their own standard curve due to potential variations in technique, incubation time, or environmental conditions. Always follow good laboratory practices when handling biological materials.
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