**Human α-CGRP ELISA Kit – For Quantitative In Vitro Determination of Human α-Calcitonin Gene-Related Peptide Concentrations in Serum, Plasma, Cerebrospinal Fluid, Tissue Homogenate, and Other Biological Fluids**
**For Laboratory Research Use Only. Not Intended for Diagnostic or Therapeutic Purposes.**
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### **INTENDED USE AND TEST PRINCIPLE**
This α-CGRP ELISA Kit is designed for laboratory research purposes only and should not be used in diagnostic or therapeutic procedures. The assay is based on the enzyme-linked immunosorbent assay (ELISA) principle, where a color change occurs upon addition of the stop solution, shifting from blue to yellow. The optical density (OD) is measured at 450 nm using a spectrophotometer.
To determine the concentration of α-CGRP in the sample, a set of calibration standards is included in the kit. These standards are run alongside the samples, allowing the operator to generate a standard curve by plotting OD values against known concentrations. The concentration of α-CGRP in the test samples is then calculated by comparing their OD values to this standard curve.
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### **SAMPLE COLLECTION AND STORAGE**
- **Serum**: Collect using a serum separator tube. Allow the sample 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 at 2000 ×g, 2–8°C. Avoid freezing and thawing.
- **Cell Culture Supernatant, Tissue Homogenate, and Other Biological Fluids**: Centrifuge to remove particulates, then analyze immediately or store at -20°C.
- **Note**: Ensure proper centrifugation, and avoid hemolysis or granulation in the samples.
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### **MATERIALS REQUIRED BUT NOT SUPPLIED**
1. 37°C incubator
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 are stored at 2–8°C. Check the expiration date on the label.
| 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 pg/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. Standards, conjugates, and plates are matched 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. Always use fresh pipette tips for each step.
5. Handle disposable plasma as potentially hazardous. Wear gloves during the procedure.
6. Dispose of all samples properly after inactivation for at least 30 minutes.
7. Avoid contact with strong acids or sodium hypochlorite.
8. Keep Substrate B away from heat or flame due to its 20% acetone content.
9. Store unused strips in sealed bags with desiccant at 2–8°C.
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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. Run standards and samples in duplicate.
2. Add 50 µL of standard or sample to appropriate wells. Blank well: 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 microtiter plate 4 times.
- **Manual Washing**: Aspirate contents, fill with 1X Wash Solution, aspirate again. Repeat 4 times.
- **Automated Washing**: Aspirate, wash 4 times with 350 µL/well.
5. After final wash, invert and blot dry.
6. Add 50 µL of Chromogen A and 50 µL of Chromogen B to each well. Mix gently and incubate for 15 minutes at 37°C, protected from light.
7. Add 50 µL of Stop Solution to each well. Color changes from blue to yellow. If uneven, tap gently.
8. Measure OD at 450 nm.
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### **DATA ANALYSIS**
1. Calculate mean OD for each standard and sample. Subtract blank OD from all values.
2. Plot standard curve (OD vs. concentration).
3. Determine sample concentration by matching OD to the curve.
4. Each user should generate their own standard curve.
5. Intra-assay CV <15%, Inter-assay CV <15%.
6. Assay range: 6.25–200 pg/mL.
7. Sensitivity: <1.0 pg/mL.
8. Cross-reactivity: No significant cross-reactivity with other peptides.
9. Storage: 2–8°C (frequent use), 6 months at -20°C.
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### **NOTES**
- Follow all safety protocols.
- Maintain accurate records of all steps.
- This kit is intended for research use only.
- Always refer to the manufacturer’s instructions for full details.
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