**Human FcγRI ELISA Kit – For the Quantitative In Vitro Determination of Human Receptor I for the Fc Region of Immunoglobulin G in Serum, Plasma, Cerebrospinal Fluid, Tissue Homogenate, and Body Fluids**
*For Laboratory Research Use Only. Not Intended for Diagnostic Procedures.*
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### **INTENDED USE AND TEST PRINCIPLE**
This Human FcγRI ELISA Kit is designed for research purposes only and is not suitable for diagnostic or therapeutic use. The assay is based on a competitive enzyme-linked immunosorbent assay (ELISA) principle. The color change from blue to yellow occurs upon addition of the Stop Solution, and the optical density (OD) is measured at 450 nm using a spectrophotometer.
To determine the concentration of FcγRI in the sample, a set of calibration standards is included in the kit. These standards are run alongside the samples to generate a standard curve, which allows for accurate quantification of FcγRI levels in the test samples by comparing their OD values to the 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. Centrifuge at 2000×g for 20 minutes. Remove 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. Store at -20°C. Avoid repeated freeze-thaw.
- **Cell Culture Supernatants, Tissue Homogenates, and Other Biological Fluids:** Centrifuge to remove particulates. Analyze immediately or store at -20°C. Avoid repeated freeze-thaw. Ensure no hemolysis or granules are present 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 (Storage: 2–8°C)**
| Name | 96 Determinations | 48 Determinations |
|------|-------------------|-------------------|
| Microtiter 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 |
*Note: Standard concentrations are 40, 20, 10, 5, 2.5, and 1.25 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. Standards, conjugate, and microtiter plates are matched for optimal performance.
2. Allow all reagents and materials to reach room temperature (20–25°C) before use. Avoid using water baths for thawing.
3. Do not use reagents beyond their expiration date.
4. Use only deionized or distilled water for dilutions.
5. Keep microtiter plate in its sealed bag until needed. Unused strips should be stored at 2–8°C with desiccant.
6. Use fresh disposable pipettes and avoid contact with strong acids or sodium hypochlorite.
7. Handle all biological fluids as potentially hazardous. Wear gloves during the procedure.
8. Allow liquid waste to stand for at least 30 minutes to inactivate viruses before disposal.
9. Substrate Solution A may be contaminated if it appears bluish; discard if so.
10. Substrate B contains 20% acetone—keep away from heat or flame.
11. Remove all kit reagents from the refrigerator and allow them to reach 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 one month.
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### **ASSAY PROCEDURE**
1. Prepare all reagents before starting. It is recommended to run each standard and sample in duplicate.
2. Add 50 µL of standard or sample to the appropriate wells. The blank well receives no sample.
3. Add 100 µL of HRP-Conjugate Reagent to all wells except the blank. Cover with an adhesive strip and incubate for 60 minutes at 37°C.
4. Wash the microtiter plate 4 times:
- **Manual Washing:** Aspirate plate contents into a waste container. Fill each well with 1X Wash Solution and aspirate again. Repeat four times.
- **Automated Washing:** Adjust washer to aspirate as much liquid as possible, with a fill volume of 350 µL per well.
5. After washing, 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, protecting from light.
6. Add 50 µL of Stop Solution. The color will turn from blue to yellow. If green or uneven, gently tap the plate to ensure mixing.
7. Read the OD at 450 nm. Generate a standard curve by plotting average OD values against corresponding FcγRI concentrations.
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### **DATA ANALYSIS**
1. Calculate the mean OD value for each standard and sample.
2. Subtract the blank OD from all readings.
3. Locate the OD value on the Y-axis and draw a horizontal line to intersect the standard curve. The corresponding X-axis value gives the FcγRI concentration.
4. Variations in technique, incubation time, or kit age can affect results. Each user should create their own standard curve.
5. Intra-assay and inter-assay CV% are less than 15%.
6. Assay range: 12.5 ng/mL – 40 ng/mL.
7. Sensitivity: <1.0 ng/mL.
8. Cross-reactivity: No significant cross-reactivity observed with recombinant or natural Human FcγRI.
9. Storage: 2–8°C (for frequent use), or -20°C (for long-term storage).
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### **NOTES**
- Always follow good laboratory practices when handling biological samples.
- This kit is intended for research use only and should not be used for clinical diagnosis.
- Ensure all steps are performed carefully to maintain accuracy and reproducibility.
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