**Human Zonulin ELISA Kit – For Quantitative In Vitro Determination of Human Zonulin in Serum, Plasma, Cerebrospinal Fluid, Tissue Homogenate, and Other Body Fluids**
*For Laboratory Research Use Only. Not for Diagnostic or Therapeutic Purposes.*
Before using this product, please read the entire package insert carefully. This ELISA kit is designed to quantitatively measure human zonulin levels in various biological samples through an enzyme-linked immunosorbent assay (ELISA). The principle of the test involves the use of a standard curve generated from known concentrations of zonulin. By comparing the optical density (OD) of the sample to this curve, the exact concentration of zonulin can be determined.
**Intended Use and Test Principle**
The Human Zonulin ELISA Kit is intended solely for laboratory research purposes and is not suitable for diagnostic or clinical use. The color change from blue to yellow upon addition of the stop solution indicates the reaction has reached completion. Calibration standards are included to ensure accurate quantification of zonulin in the samples.
**Sample Collection and Storage**
- **Serum**: Collect using a serum separator tube. Allow clotting for 2 hours at room temperature or overnight at 4°C. Centrifuge at 2000×g for 20 minutes. Store at -20°C, avoiding repeated freeze-thaw cycles.
- **Plasma**: Use heparin as an anticoagulant. Centrifuge within 30 minutes at 2000×g (2–8°C). Store at -20°C.
- **Cell Culture Supernatants, Tissue Homogenates, and Other Biological Fluids**: Centrifuge to remove particulates. Assay immediately or store at -20°C. Ensure no hemolysis or granules are present in the samples.
**Materials Required but Not Supplied**
- 37°C incubator
- Microplate reader capable of measuring absorbance at 450 nm
- Precision pipettes, disposable tips, and absorbent paper
- Distilled or deionized water
**Reagents Provided**
All reagents should be stored at 2–8°C. Refer to the expiration date on the label.
- Microtiter Strip Plate (12×8 or 12×4 strips)
- Standards (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 Solutions A & B (6.0 ml or 3.0 ml each)
- Stop Solution (6.0 ml or 3.0 ml)
- Closure Plate Membrane (2 units)
- User Manual (1 copy)
- Sealed Bags (1 per kit)
**Precautions**
- Do not mix reagents from different kits. Each kit is calibrated for optimal performance.
- Allow all reagents and samples to reach room temperature before use. Avoid using water baths for thawing.
- Do not use reagents past their expiration date.
- Use only deionized or distilled water for dilutions.
- Keep microtiter plates in their sealed bags until needed. Unused strips should be stored with desiccant.
- Use fresh pipette tips for each transfer to prevent contamination.
- All blood-derived products should be treated as potentially infectious. Follow proper biosafety protocols.
- Dispose of waste properly, including inactivation with hypochlorite at 1.0% concentration for 30 minutes.
- Avoid contamination of substrate solutions.
- Substrate B contains 20% acetone—keep away from heat or flame.
**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 blank. Cover with adhesive strip and incubate for 60 minutes at 37°C.
4. Wash microtiter plate 4 times. Manual washing involves filling and aspirating each well with 1X wash solution. Automated washing requires 4 cycles 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 uneven, gently tap the plate.
7. Read OD at 450 nm. Plot standard curve using average OD values. Calculate sample concentrations by matching OD to the curve.
**Performance Characteristics**
- Intra-assay and Inter-assay CV < 15%.
- Assay range: 62.5 ng/mL – 2000 ng/mL.
- Sensitivity: <10 ng/mL.
- Cross-reactivity: No significant cross-reactivity with other proteins.
- Storage: 2–8°C (frequent use), 6 months at -20°C.
**Important Notes**
- Always follow safety guidelines when handling biological samples.
- Ensure all steps are performed accurately to maintain data reliability.
- Each user should generate their own standard curve for best results.
**Quote:** "Accuracy and consistency are key in every step of the ELISA process."
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