**Human FVII ELISA Kit – For the Quantitative In Vitro Determination of Human Coagulation Factor VII 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 kit is designed for research purposes only and should not be used in clinical diagnostic settings.
**INTENDED USE AND TEST PRINCIPLE**
The Human FVII ELISA Kit is intended for laboratory research use only and is not suitable for diagnostic or therapeutic applications. The assay is based on a competitive enzyme-linked immunosorbent assay (ELISA) principle. The stop solution changes the color from blue to yellow, and the intensity of the color is proportional to the concentration of FVII in the sample.
To determine the FVII concentration, a set of calibration standards is included. These standards are run alongside the samples to generate a standard curve by plotting optical density (OD) values against known concentrations. The FVII levels in the test samples are then calculated by comparing their OD values to the standard curve.
**SAMPLE COLLECTION AND STORAGE**
- **Serum**: Collect using a serum separator tube. Allow the blood to clot for 2 hours at room temperature or overnight at 4°C. Centrifuge for 20 minutes at ~2000×g. Remove the serum and assay immediately, or aliquot and 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. Avoid freeze-thaw cycles.
- **Cell Culture Supernatants, Tissue Homogenates, and Other Biological Fluids**: Centrifuge to remove particulates and assay immediately, or aliquot and store at -20°C. Ensure samples are properly centrifuged, and avoid hemolysis or contamination with granules.
**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
**REAGENTS PROVIDED**
All reagents should be stored at 2–8°C. Check the expiration date on the label before use.
| Reagent | 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 are 40, 20, 10, 5, 2.5, and 1.25 IU/ml.*
**PRECAUTIONS AND GUIDELINES**
1. If sample values exceed the highest standard, dilute with sample diluent and repeat the assay.
2. Only use kit reagents as specified. Do not mix components from different kits.
3. Allow all reagents and samples to reach room temperature (20–25°C) before use. Do not thaw using water baths.
4. Do not use any reagents beyond their expiration date.
5. Use only deionized or distilled water for dilutions.
6. Keep microtiter plates in sealed bags until needed. Unused strips must be stored with desiccant at 2–8°C.
7. Use fresh pipette tips for each transfer to prevent cross-contamination.
8. All biological materials should be treated as potentially infectious. Follow proper biosafety protocols.
9. Dispose of all waste according to local regulations.
10. Liquid waste: Add sodium hypochlorite to a final concentration of 1.0% and let stand for at least 30 minutes before disposal.
11. Substrate solutions are sensitive to contamination. If the solution appears bluish, discard it.
12. Chromogen B contains 20% acetone; keep away from heat and open flames.
13. Allow all reagents to reach room temperature before use.
**REAGENT PREPARATION**
- **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. Run standards and samples in duplicate.
2. Add 50 μl of standard or sample to appropriate wells. 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 plate four times.
- **Manual Washing**: Aspirate contents, fill with 1X Wash Solution, aspirate again. Repeat four times. Invert and blot dry.
- **Automated Washing**: Aspirate and wash four times with 1X Washer Buffer. Adjust brush to remove maximum liquid. Fill 350 μL/well.
5. 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.
6. Add 50 μl of Stop Solution to each well. Read OD at 450 nm.
**DATA ANALYSIS**
1. Plot the average OD values of the six standards on the Y-axis versus concentration on the X-axis.
2. Calculate the mean OD for each standard and sample. Subtract the blank OD value from all readings.
3. Generate the standard curve using graphing software.
4. Locate the OD value of each sample on the Y-axis and draw a horizontal line to intersect the curve. Draw a vertical line to the X-axis to determine the FVII concentration.
5. Intra-assay and inter-assay CV% are less than 15%.
6. Assay range: 12.5 – 40 IU/ml.
7. Sensitivity: <1.0 IU/ml.
8. No significant cross-reactivity or interference observed.
9. Storage: 2–8°C for frequent use; -20°C for long-term storage (up to 6 months).
**NOTES**
This kit is intended for research use only. Always follow safety guidelines when handling biological materials. For detailed instructions, refer to the complete user manual provided.
Lifting Columns
Suzhou Herstar Medical Technology Co., Ltd. , https://www.hosunherstar.com