**Human FcγRIIb ELISA Kit – For the quantitative in vitro determination of Human Receptor IIb for the Fc region of immunoglobulin G concentrations in serum, plasma, cerebrospinal fluid, tissue homogenate, and other body fluids. Intended for laboratory research use only. NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES.**
Before using this product, please read the entire instruction manual carefully.
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### **INTENDED USE AND TEST PRINCIPLE**
This Human FcγRIIb ELISA Kit is designed for laboratory research purposes only and should not be used for diagnostic or therapeutic applications. The kit is based on a sandwich ELISA principle, where the target protein (FcγRIIb) in the sample binds to immobilized antibodies on the microplate. A horseradish peroxidase (HRP)-conjugated secondary antibody is then added, followed by a chromogenic substrate that produces a color change proportional to the amount of FcγRIIb present. The reaction is stopped, and the optical density (OD) is measured at 450 nm. A standard curve is generated using known concentrations of FcγRIIb, allowing for accurate quantification of unknown samples.
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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. Centrifuge at 2000×g for 20 minutes. Remove serum and assay 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 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. Avoid repeated freezing and thawing.
*Note: Ensure adequate centrifugation and avoid hemolysis or granulation in 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 are 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 |
*Standard concentrations: 100, 50, 25, 12.5, 6.25, 3.12 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, conjugates, and plates are matched for optimal performance. Only use reagents provided by the manufacturer.
2. Allow all reagents and materials to reach room temperature (20–25°C) before use. Do not use water baths to thaw samples or reagents.
3. Do not use reagents beyond their expiration date.
4. Use only deionized or distilled water for dilutions.
5. Keep microtiter plates in their sealed bags until needed. Unused strips should be stored at 2–8°C with desiccant.
6. Wear gloves during handling. All blood-derived products should be treated as potentially infectious.
7. Waste should be inactivated for at least 30 minutes before disposal.
8. Substrate B contains 20% acetone; keep away from heat and flame.
9. Allow all reagents to warm up to room temperature before starting the assay.
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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 standards and samples in duplicate.
2. Add 50 µL of standard or sample to the appropriate wells.
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 plate 4 times.
- **Manual Washing**: Aspirate contents, fill each well with 1X Wash Solution, and aspirate again. Repeat 4 times.
- **Automated Washing**: Aspirate and wash 4 times with 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. The color should turn yellow. If green or uneven, gently tap the plate.
7. Read OD at 450 nm within 15 minutes.
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### **CALCULATION OF RESULTS**
1. Plot the average OD (450 nm) of each standard against its concentration to create a standard curve.
2. Subtract the blank OD from all readings.
3. Locate the OD value of each sample on the Y-axis and draw a horizontal line to intersect the standard curve. Determine the corresponding concentration.
4. Variability may occur due to operator technique, pipetting, washing, incubation time, or kit age. Each user should generate their own standard curve.
5. Intra-assay and inter-assay CV% are <15%.
6. Assay range: 3.12 – 100 ng/mL
7. Sensitivity: <1.0 ng/mL
8. Cross-reactivity: No significant cross-reaction with other proteins.
9. Storage: 2–8°C (frequent use); 6 months at -20°C.
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**Please follow all instructions carefully. This kit is not intended for clinical use.**
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