**Human FETU-B ELISA Kit – For the Quantitative In Vitro Determination of Human FETU-B Concentrations in Serum, Plasma, Cerebrospinal Fluid, Tissue Homogenate, and Other Body Fluids**
**For Laboratory Research Use Only. Not for Diagnostic or Therapeutic Procedures.**
This ELISA kit is designed for the quantitative measurement of Human FETU-B levels in various biological samples. The assay is based on a competitive immunoassay principle, where the color change resulting from the reaction is measured at 450 nm using a microplate reader. A standard curve is generated using known concentrations of FETU-B, allowing the accurate determination of unknown sample concentrations.
**Intended Use and Test Principle**
The Human FETU-B ELISA Kit is intended for laboratory research use only and should not be used in diagnostic or therapeutic procedures. The test involves binding of FETU-B to specific antibodies coated on the microtiter plate. After incubation, the enzyme-linked HRP-conjugated antibody binds to the complex, and the chromogen solution produces a color change that is proportional to the amount of FETU-B present. The Stop Solution halts the reaction, and the optical density (OD) is measured at 450 nm. By comparing the OD values of the samples to the standard curve, the concentration of FETU-B can be accurately determined.
**Sample Collection and Storage**
- **Serum**: Use serum separator tubes. Allow samples to clot for 2 hours at room temperature or overnight at 4°C before centrifugation at 2000×g for 20 minutes. Remove serum and assay immediately or 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 and assay immediately or store at -20°C. Ensure no hemolysis or granules are present.
**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 (Stored at 2–8°C)**
- MicroELISA Strip Plate: 12×8 strips (96 determinations), 12×4 strips (48 determinations)
- Standards (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 per kit
**Standard Concentrations**: 1000, 500, 250, 125, 62.5, 31.2 μg/mL
**Important Notes**
- Always allow reagents and samples to reach room temperature (20–25°C) before use.
- Do not use water baths for thawing.
- Do not use reagents beyond their expiration date.
- Use only deionized or distilled water for dilutions.
- Keep unused strip wells in sealed bags with desiccant.
- Use fresh pipette tips for each transfer to avoid contamination.
**Safety Information**
All samples should be treated as potentially infectious. Follow proper biosafety protocols when handling biological materials. Disposable items must be disposed of properly.
**Wash Solution Preparation**
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.
2. Add 50 µL of standards or samples to appropriate wells (excluding blank). Cover with adhesive strip and incubate for 60 minutes at 37°C.
3. Wash the microtiter plate 4 times using either manual or automated washing.
4. Add 50 µL of Chromogen A and 50 µL of Chromogen B to each well. Incubate for 15 minutes at 37°C, protected from light.
5. Add 50 µL of Stop Solution to each well. Read OD at 450 nm.
6. Plot the average OD values of standards against their concentrations to generate a standard curve. Determine sample concentrations by comparing their OD values to the curve.
**Performance Characteristics**
- Intra-assay CV (%) and Inter-assay CV (%) < 15%
- Assay range: 31.2 – 1000 μg/mL
- Sensitivity: <10 μg/mL
- Cross-reactivity: No significant cross-reactivity observed
- Storage: 2–8°C (frequent use); -20°C (long-term storage)
**Note**: This product is intended for research purposes only. It is not approved for clinical diagnostics. Always read the entire package insert before use.
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