**Human SEMA3A ELISA Kit – For the Quantitative In Vitro Determination of Human Semaphorin 3A 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 is not intended for clinical diagnosis.
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### **INTENDED USE AND TEST PRINCIPLE**
This Human SEMA3A ELISA Kit is specifically developed for the quantitative determination of Semaphorin 3A (SEMA3A) concentrations in biological samples such as serum, plasma, cerebrospinal fluid, tissue homogenates, and other body fluids. The test is based on the enzyme-linked immunosorbent assay (ELISA) principle, where a colorimetric reaction is used to detect and quantify SEMA3A levels.
The kit includes a set of calibration standards that are run alongside the samples. By plotting the optical density (OD) values against known SEMA3A concentrations, a standard curve is generated. The sample concentrations are then calculated by comparing their OD readings to this curve.
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### **SAMPLE COLLECTION AND STORAGE**
- **Serum**: Collect using a serum separator tube. Allow samples to clot for 2 hours at room temperature or overnight at 4°C before centrifuging at 2000×g for 20 minutes. Remove the serum and analyze immediately, or aliquot and 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. Avoid repeated freezing and thawing.
- **Cell Culture Supernatants, Tissue Homogenates, and Other Biological Fluids**: Centrifuge to remove particulates, and analyze immediately or store at -20°C. Ensure no hemolysis or granules are present in the samples.
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### **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
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### **REAGENTS PROVIDED**
| Reagent | 96 Determinations | 48 Determinations |
|--------|-------------------|-------------------|
| MicroElisa Stripplate | 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: 200, 100, 50, 25, 12.5, 6.25 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 substitute reagents from different kit lots. All components are calibrated for optimal performance.
2. Allow all reagents and materials to reach room temperature (20–25°C) before use. Do not use water baths for thawing.
3. Do not use any reagents past their expiration date.
4. Use only deionized or distilled water for dilutions.
5. Keep unused microtiter plate strips in their sealed pouch with desiccant.
6. Always use fresh disposable pipette tips to avoid contamination.
7. Treat all blood-derived products as potentially infectious. Follow proper biosafety protocols.
8. Dispose of all samples and waste according to local regulations.
9. Liquid waste should be treated with sodium hypochlorite (1.0% final concentration) for at least 30 minutes before disposal.
10. Substrate solutions may be easily contaminated. Handle with care.
11. Chromogen B contains 20% acetone—keep away from heat and flame.
12. Allow all reagents to warm to room temperature before use.
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### **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 1 month.
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### **ASSAY PROCEDURE**
1. Prepare all reagents before starting. Run standards and samples in duplicate.
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.
4. Wash the plate four times with 1X Wash Solution. After the final wash, invert and blot dry.
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. The color will change from blue to yellow.
7. Measure OD at 450 nm using a microplate reader.
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### **DATA ANALYSIS**
1. Plot the average OD values of the standards against their concentrations to generate a standard curve.
2. Subtract the blank OD from all sample OD values before interpretation.
3. Locate the sample OD on the Y-axis and draw a horizontal line to intersect the standard curve. Read the corresponding SEMA3A concentration from the X-axis.
4. Each user should generate their own standard curve due to potential variations in technique.
5. Intra-assay CV <15%, Inter-assay CV <15%.
6. Assay range: 6.25–200 ng/mL
7. Sensitivity: <1.0 ng/mL
8. Cross-reactivity: No significant cross-reactivity observed with other proteins.
9. Storage: 2–8°C (for frequent use); -20°C for long-term storage (up to 6 months).
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**Important Note:** This kit is for research use only. It is not approved for diagnostic or clinical applications. Always follow proper safety procedures when handling biological samples and reagents.
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