**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. Intended for laboratory research use only. NOT FOR DIAGNOSTIC OR THERAPEUTIC USE.**
Before using this kit, please read the entire package insert carefully.
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### **INTENDED USE AND TEST PRINCIPLE**
This Human SEMA3A ELISA Kit is designed for research purposes only and is not intended for diagnostic or therapeutic procedures. The kit allows for the accurate quantification of SEMA3A levels in various biological samples through a colorimetric enzyme-linked immunosorbent assay (ELISA).
The principle of the assay relies on a standard curve. Calibration standards are used alongside the test samples to generate a relationship between optical density (OD) readings and SEMA3A concentration. By comparing the OD values of the samples to this standard curve, the concentration of SEMA3A in each sample can be accurately determined.
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### **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 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 and avoid freeze-thaw cycles.
- **Cell culture supernatants, tissue homogenates, and other body fluids**: Centrifuge to remove particulates and analyze immediately, or aliquot and 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 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
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### **REAGENTS PROVIDED**
All reagents should be stored at 2–8°C. Check the expiration date on the label before use.
| Reagent 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: 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 mix reagents from different kit lots. All components are calibrated for optimal performance.
2. Allow all reagents and samples to reach room temperature (20–25°C) before use. Do not thaw using water baths.
3. Do not use reagents beyond their expiration date.
4. Only use deionized or distilled water for dilutions.
5. Keep microtiter plates in their sealed bags until ready to use. Unused strips must be stored with desiccant at 2–8°C.
6. Use fresh pipette tips for each transfer to prevent cross-contamination.
7. Disposable materials must be treated as potentially hazardous and disposed of accordingly.
8. Always follow biosafety protocols when handling biological samples.
9. Dispose of all waste properly, including liquid waste, by adding 1% sodium hypochlorite and allowing it to sit for at least 30 minutes before disposal.
10. Substrate solutions may be easily contaminated. Avoid exposure to heat or flame, especially for Solution B containing 20% acetone.
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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 one month.
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### **ASSAY PROCEDURE**
1. Prepare all reagents before starting the assay. It is recommended to run standards and samples in duplicate.
2. Add 50 µL of standard or sample to appropriate wells. The blank well receives 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 the plate and blot dry with absorbent paper.
- *Automated washing:* Aspirate all wells, then wash four times with 1X Wash Buffer. Adjust aspiration volume to 350 µL per well.
5. Add 50 µL of Chromogen Solution A and 50 µL of Chromogen Solution B to each well. Mix gently and incubate for 15 minutes at 37°C, away from light.
6. Add 50 µL of Stop Solution to each well. The color should change from blue to yellow. If uneven, gently tap the plate.
7. Measure OD at 450 nm using a microplate reader.
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### **DATA ANALYSIS**
1. Plot the average OD (450 nm) of each standard against its concentration to create a standard curve.
2. Calculate the mean OD value for each standard and sample. Subtract the blank OD from all readings.
3. Locate the sample OD on the Y-axis and draw a horizontal line to intersect the standard curve. Then draw a vertical line to the X-axis to determine the SEMA3A concentration.
4. Variability due to operator technique, pipetting, incubation time, or kit age may affect results. Each user should generate their own standard curve.
5. Intra-assay and inter-assay CV% are less than 15%.
6. Assay range: 6.25 ng/mL to 200 ng/mL.
7. Sensitivity: <1.0 ng/mL.
8. Cross-reactivity: No significant cross-reactivity with other proteins.
9. Storage: 2–8°C for frequent use; -20°C for long-term storage (up to 6 months).
10. **Always refer to the manufacturer’s instructions for complete details.**
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