**Human FcεRII ELISA Kit – For the quantitative in vitro determination of Human Receptor II for the Fc region of immunoglobulin E concentrations in serum, plasma, cerebrospinal fluid, tissue homogenate, and other body fluids. FOR LABORATORY RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.**
This ELISA kit is designed to measure the concentration of FcεRII in various biological samples. The kit includes a set of calibration standards that are run alongside the samples to generate a standard curve. By comparing the optical density (OD) values of the samples to this curve, the exact concentration of FcεRII can be determined.
**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 at 2000×g for 20 minutes. 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. Avoid repeated 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 free from hemolysis or granules.
**Materials Required but Not Supplied:**
1. Incubator 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 (Stored at 2–8°C):**
- MicroELISA Strip Plate (12×8 strips / 12×4 strips)
- Standard (6 vials, 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 units)
- User Manual (1 copy)
- Sealed Bags (1 unit)
**Note:** Standard concentrations are 20, 10, 5, 2.5, 1.25, and 0.625 ng/ml. If sample values exceed the highest standard, dilute with sample diluent and repeat the assay.
**Precautions:**
- Do not mix reagents from different kits. All components are calibrated for optimal performance.
- Allow all reagents and samples to reach room temperature (20–25°C) before use.
- Do not use reagents past their expiration date.
- Use only deionized or distilled water for dilution.
- Keep unused microtiter plate strips in the sealed bag with desiccant until needed.
- Wear gloves during the procedure. Handle all biological materials as potentially infectious.
- Dispose of samples and waste according to local regulations. Add 1% sodium hypochlorite to liquid waste and let stand for 30 minutes before disposal.
- Avoid contamination of substrate solutions. Discard if discolored.
- Store all reagents at 2–8°C for frequent use, or at -20°C for long-term storage (up to six months).
**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. 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 microtiter plate four times. Manual washing involves aspirating and refilling with wash solution. Automated washing should be done with 350 μL per well.
5. Add 50 μl each of Chromogen A and B to each well. Incubate for 15 minutes at 37°C, protected from light.
6. Add 50 μl of Stop Solution. The color changes from blue to yellow. If uneven, gently tap the plate.
7. Measure OD at 450 nm. Plot the average OD values of the standards against their concentrations to create a standard curve.
**Interpretation:**
- Calculate mean OD for each standard and sample. Subtract blank OD value from all readings.
- Locate the OD on the Y-axis and draw a horizontal line to the curve. Read the corresponding concentration.
- Intra-assay and inter-assay CVs are less than 15%.
**Assay Range:** 0.625 ng/ml – 20 ng/ml
**Sensitivity:** < 0.1 ng/ml
**Cross-reactivity:** No significant cross-reactivity observed. Recognizes both recombinant and natural human FcεRII.
**Storage:** 2–8°C for frequent use; -20°C for long-term storage (up to six months).
**Note:** Always read the full user manual before performing the assay.
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