**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 Use in Diagnostic Procedures.**
This package insert must be read carefully before using the Mouse Histamine ELISA Kit. The kit is designed for research purposes only and is not intended for diagnostic or therapeutic use. The principle of the assay is based on a competitive enzyme-linked immunosorbent assay (ELISA), where histamine in the sample competes with a histamine-enzyme conjugate for binding to specific antibodies coated on the microtiter plate. After incubation, the Stop Solution changes the color from blue to yellow, and the optical density (OD) is measured at 450 nm using a microplate reader. A standard curve is generated using known concentrations of histamine, allowing the quantification of unknown samples by comparison.
**Sample Collection and Storage:**
- **Serum:** Use 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:** Collect using 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 and store at -20°C. Ensure no hemolysis or contamination occurs.
**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):**
- Microplate: 12×8 strips (96 determinations) / 12×4 strips (48 determinations)
- 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. All components are matched for optimal performance.
- Allow all reagents and materials to reach room temperature (20–25°C) before use.
- Do not thaw samples or reagents using a water bath.
- Avoid using expired reagents.
- Always use deionized or distilled water for dilutions.
- Keep unused microplate strips in their sealed bag with desiccant.
- Use fresh pipette tips for each transfer to prevent cross-contamination.
- Wear disposable gloves throughout the procedure.
- Treat all biological samples as potentially infectious.
- Dispose of waste properly, including liquid waste treated with 1% sodium hypochlorite.
- Avoid exposure of Substrate B to heat or flame due to its acetone content.
- Ensure all reagents are at room temperature before starting the assay.
**Assay Procedure:**
1. Prepare all reagents and add them to the microplate.
2. Incubate for 60 minutes at 37°C.
3. Wash the plate 4 times using 1X Wash Solution.
4. Add 50 µL of Chromogen A and B to each well. Incubate for 15 minutes at 37°C.
5. Add 50 µL of Stop Solution. Read OD at 450 nm within 15 minutes.
**Data Analysis:**
- Plot average OD values of standards vs. concentration.
- Subtract blank OD from all readings.
- Determine sample concentration by comparing OD to the standard curve.
- Intra-assay CV <15%, Inter-assay CV <15%.
- Assay range: 1.0–32 ng/mL.
- Sensitivity: <0.5 ng/mL.
- No significant cross-reactivity with other substances.
**Storage:**
- Store at 2–8°C for frequent use.
- For long-term storage, keep at -20°C for up to 6 months.
**Note:** Always follow good laboratory practices and safety protocols when handling biological samples and reagents.
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