**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 PROCEDURES.**
Before using this product, please read the entire insert carefully. This ELISA kit is designed to measure the levels of FcγRIIb in various biological samples. The test is based on a sandwich immunoassay principle, where the FcγRIIb in the sample binds to the immobilized antibodies on the microtiter plate. A horseradish peroxidase (HRP)-conjugated secondary antibody is then added, followed by a chromogenic substrate that produces a color change proportional to the concentration of FcγRIIb in the sample. The reaction is stopped, and the optical density (OD) is measured at 450 nm.
**INTENDED USE AND TEST PRINCIPLE**
This FcγRIIb ELISA Kit is intended for laboratory research use only and should not be used for diagnostic or therapeutic purposes. The Stop Solution changes the color from blue to yellow, indicating the end of the reaction. The calibration standards are run alongside the samples to generate a standard curve, which allows for accurate quantification of FcγRIIb in unknown samples.
**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 (20 minutes at ~2000×g). Remove the 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.
- **Cell culture supernatants, tissue homogenates, and other body fluids**: Centrifuge to remove particulates. Assay immediately or store at -20°C. Avoid repeated freezing and thawing.
*Note: Ensure adequate centrifugation and avoid hemolysis or granulation in the samples.*
**MATERIALS REQUIRED BUT NOT SUPPLIED**
1. 37°C incubator
2. Standard 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 are stored at 2–8°C. Check the expiration date on the label.
| Name | 96 determinations | 48 determinations |
|------|-------------------|-------------------|
| MicroELISA Stripplate | 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: 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.*
**PRECAUTIONS**
1. Do not mix reagents from different kit lots. All components are matched for optimal performance. Only use reagents provided by the manufacturer.
2. Allow all reagents and samples to reach room temperature (20–25°C) before use. Do not use water baths to thaw samples.
3. Do not use reagents past their expiration date.
4. Use only deionized or distilled water for dilutions.
5. Keep microtiter plates in sealed bags until use. Unused strips should be stored at 2–8°C with desiccant.
6. Wear gloves during the procedure. Handle all biological samples as potentially infectious.
7. Waste should be inactivated for 30 minutes before disposal.
8. Chromogen Solution B contains 20% acetone—keep away from heat or flame.
9. Allow all reagents to reach room temperature before use.
**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.
**ASSAY PROCEDURE**
1. Prepare all reagents before starting. Add Standards and Samples in duplicate to the microtiter plate.
2. Add 50 µl of Standard or Sample to each well.
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 manually or automatically.
- *Manual*: Fill each well with 1X Wash Solution, aspirate, and repeat four times.
- *Automated*: Aspirate and wash four times with 350 µl/well.
5. Add 50 µl of Chromogen A and 50 µl of Chromogen B to each well. Incubate for 15 minutes at 37°C, protecting 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. Measure OD at 450 nm within 15 minutes.
**CALCULATION OF RESULTS**
1. Plot the average OD values (450 nm) against standard concentrations to create a standard curve.
2. Subtract the blank well OD from all measurements.
3. Locate the OD value on the Y-axis and find the corresponding concentration on the X-axis.
4. Each user should generate their own standard curve.
5. Intra-assay and inter-assay CVs are less than 15%.
6. Assay range: 3.12 ng/ml – 100 ng/ml.
7. Sensitivity: <1.0 ng/ml.
8. No significant cross-reactivity observed.
9. Storage: 2–8°C (frequent use); 6 months at -20°C.
**NOTE:** Always follow good laboratory practices. Handle all reagents and samples with care. This kit is not suitable for clinical use.
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