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Human FGF-4 ELISA Kit
**Human FGF-4 ELISA Kit – For the quantitative in vitro determination of Human fibroblast growth factor-4 (FGF-4) 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 kit, please read the entire package insert carefully. This ELISA kit is designed to measure FGF-4 levels through a colorimetric reaction, allowing researchers to quantify the protein concentration in various biological samples.
The test works by binding FGF-4 in the sample to specific antibodies coated on a microtiter plate. After incubation, a horseradish peroxidase (HRP)-conjugated antibody is added, which binds to the captured FGF-4. A chromogenic substrate is then introduced, causing a color change that correlates with the amount of FGF-4 present. The optical density (OD) is measured at 450 nm, and the concentration is determined by comparing the sample OD to a standard curve generated from known concentrations of FGF-4.
**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 freezing.
- **Cell culture supernatants, tissue homogenates, and other biological fluids:** Centrifuge to remove particulates, then assay immediately or store at -20°C. Ensure no hemolysis or contamination occurs.
**Materials Required (Not Included):**
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 (Store at 2–8°C):**
- MicroELISA strip plate (12×8 or 12×4 strips)
- Standard (6 vials, 0.5 mL/vial)
- Sample Diluent (6.0 mL or 3.0 mL)
- HRP-Conjugate Reagent (10.0 mL or 5.0 mL)
- 20X Wash Solution (25 mL or 15 mL)
- Chromogen Solution A (6.0 mL or 3.0 mL)
- Chromogen Solution B (6.0 mL or 3.0 mL)
- Stop Solution (6.0 mL or 3.0 mL)
- Closure Plate Membrane (2 pieces)
- User Manual (1 copy)
- Sealed Bags (1 each)
**Precautions:**
- Do not mix reagents from different kits. Each component is calibrated for optimal performance.
- Avoid thawing samples or reagents in water baths.
- Do not use reagents beyond their expiration date.
- Only use deionized or distilled water for dilution.
- Keep unused microplate strips in the sealed bag with desiccant.
- Use fresh pipette tips for each transfer to prevent cross-contamination.
- Never mix acid and sodium hypochlorite.
- Handle all samples as potentially infectious. Wear gloves during the procedure.
- Dispose of all waste properly, including liquid waste treated with 1% sodium hypochlorite.
- Avoid exposure of Chromogen B to heat or flame due to its acetone content.
- Allow all reagents to reach room temperature (20–25°C) before use.
**Reagent Preparation:**
- Wash Solution (1X): Dilute 1 part of 20X Wash Solution with 19 parts of deionized or distilled water. Store at 2–8°C for up to one month.
**Assay Procedure:**
1. Prepare all reagents before adding 100 µL of HRP-conjugate to standard and sample wells (excluding blank). Cover with adhesive strips and incubate for 60 minutes at 37°C.
2. Wash the microtiter plate 4 times manually or using an automated washer.
3. 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.
4. Add 50 µL of Stop Solution to each well. The color should change from blue to yellow. If uneven, gently tap the plate.
5. Measure OD at 450 nm within 15 minutes using a microplate reader.
**Calculation of Results:**
- Plot the average OD values of standards vs. their concentrations to create a standard curve.
- Subtract blank OD from all measurements.
- Determine sample concentration by matching OD to the standard curve.
- Intra-assay and inter-assay CVs are <15%. Sensitivity is <1 pg/mL.
- Cross-reactivity with other FGFs is minimal.
- Store at 2–8°C (frequent use) or -20°C (long-term storage).
**Important Notes:**
- This kit is for research use only. Not intended for diagnostic or therapeutic purposes.
- Always follow safety protocols when handling biological samples.
- Read the full procedure before starting the assay.
This product is designed for scientific research and must be used in accordance with applicable biosafety regulations.