**Mouse Histamine ELISA Kit – For the Quantitative In Vitro Determination of Mouse Histamine Concentrations in Serum, Plasma, Cerebrospinal Fluid, Tissue Homogenate, and Other Body Fluids**
**Catalogue Number: CK-E20452**
**For Laboratory Research Use Only. Not for Diagnostic or Therapeutic Procedures.**
This ELISA kit is designed for the quantitative determination of histamine (HIS) in various biological samples such as serum, plasma, cerebrospinal fluid, tissue homogenates, and other body fluids. The assay is based on a competitive enzyme-linked immunosorbent assay (ELISA) principle. A standard curve is generated using a set of calibration standards, allowing the accurate quantification of HIS concentrations in unknown samples by comparing their optical density (OD) values at 450 nm.
**Intended Use and Test Principle**
The Mouse Histamine ELISA Kit is intended for research purposes only and should not be used in diagnostic or therapeutic applications. The test involves the binding of histamine to specific antibodies coated on microtiter plates. After incubation and washing steps, a chromogenic substrate is added, producing a color change that is measured spectrophotometrically at 450 nm. The intensity of the color correlates with the concentration of histamine in the sample.
**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 after aliquoting. Avoid repeated freeze-thaw cycles.
- **Plasma**: Use 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 Biological Fluids**: Centrifuge to remove particulates and 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**
All reagents are stored at 2–8°C. Refer to the expiration date on the label.
- Microtiter 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 unit)
- Sealed Bags (1 unit)
**Important Notes**
- Standard concentrations: 32, 16, 8, 4, 2, 1 ng/mL.
- If sample values exceed the highest standard, dilute with Sample Diluent.
- Do not mix reagents from different kit lots.
- Allow all reagents to reach room temperature before use.
- Do not use expired reagents.
- Use only deionized or distilled water for dilutions.
- Keep microtiter plates in sealed bags until use.
- Use fresh pipette tips for each transfer to avoid cross-contamination.
- All samples should be handled with caution, as they may contain infectious agents.
- Dispose of waste properly. Add 1.0% sodium hypochlorite to liquid waste and allow 30 minutes for inactivation.
- Substrate solutions must be used before they turn blue.
- Chromogen 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 1 month.
**Assay Procedure**
1. Prepare all reagents and add them to the microtiter plate.
2. Incubate for 60 minutes at 37°C.
3. Wash the plate 4 times manually or automatically.
4. Add 50 µL of Chromogen A and 50 µL of Chromogen B to each well. Incubate for 15 minutes at 37°C.
5. Add 50 µL of Stop Solution. The color changes from blue to yellow.
6. Measure OD at 450 nm within 15 minutes.
**Calculation of Results**
1. Plot average OD values against standard concentrations.
2. Subtract blank OD from all readings.
3. Determine sample concentration by interpolating on the standard curve.
4. Intra-assay and inter-assay CV% are less than 15%.
5. Assay range: 1.0–32 ng/mL.
6. Sensitivity: <0.5 ng/mL.
7. Cross-reactivity: No significant interference observed.
8. Storage: 2–8°C (frequent use); -20°C for long-term storage (up to 6 months).
**Safety and Disposal**
Handle all samples and reagents with care. Follow standard laboratory safety protocols. Dispose of waste according to local regulations.
**Note:** This kit is intended for research use only. It is not approved for clinical diagnostics. Always follow the manufacturer's instructions and maintain proper lab practices.
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