**Rat Histamine ELISA Technical Note**
This reagent is intended for research use only and not for diagnostic or therapeutic purposes. The kit is designed to quantify histamine levels in rat serum, plasma, and related biological fluids.
**Principle of the Assay**
The method employed is a double-antibody sandwich ELISA. A microtiter plate is pre-coated with a purified monoclonal antibody specific to rat histamine. After adding the sample, histamine binds to the immobilized antibody. An HRP-conjugated secondary antibody is then introduced, forming a complex of antibody-antigen-enzyme-labeled antibody. Following washing steps, TMB (3,3',5,5'-tetramethylbenzidine) substrate is added. Under the catalytic action of HRP, TMB turns blue and is then converted to yellow upon acid termination. The intensity of the color is directly proportional to the histamine concentration in the sample. The optical density (OD) is measured at 450 nm using a microplate reader, and the histamine concentration is determined by comparing the OD value to a standard curve.
**Kit Components**
The kit includes:
- 1×48 or 1×96 well microtiter plate
- 2 sealing films (for 48-well configuration) or 2 (for 96-well)
- 1 standard (18 μg/L)
- 1 standard diluent (0.5 ml × 1 bottle)
- 1 enzyme-labeled reagent (3 ml × 1 bottle)
- 1 sample diluent (3 ml × 1 bottle)
- 1 developer A (3 ml × 1 bottle)
- 1 developer B (3 ml × 1 bottle)
- 1 stop solution (3 ml × 1 bottle)
- 1 concentrated wash buffer (20 ml × 20 times or 20 ml × 30 times)
All components should be stored at 2–8°C.
**Sample Preparation and Handling**
- **Serum**: Allow blood to clot at room temperature for 10–20 minutes, then centrifuge at 2000–3000 rpm for 20 minutes. Collect supernatant; if precipitate forms, re-centrifuge.
- **Plasma**: Use EDTA or sodium citrate as anticoagulant. Mix for 10–20 minutes, then centrifuge.
- **Urine**: Centrifuge at 2000–3000 rpm for 20 minutes. Collect supernatant.
- **Cell culture supernatant**: Centrifuge at 2000–3000 rpm for 20 minutes. For intracellular components, lyse cells via freeze-thaw cycles and centrifuge again.
- **Tissue samples**: Homogenize in PBS (pH 7.4), centrifuge, and collect supernatant. Store at 2–8°C after thawing.
- **Storage**: Process samples immediately after collection. If not tested right away, store at -20°C. Avoid repeated freeze-thaw cycles.
**Notes on Sample Inhibition**
Sodium azide (NaN₃) should not be present in samples, as it inhibits horseradish peroxidase (HRP).
**Procedure Summary**
1. **Standard preparation**: Prepare serial dilutions of the standard.
2. **Sample addition**: Add 40 μl sample diluent and 10 μl sample to each well (final 5-fold dilution).
3. **Incubation**: Seal the plate and incubate at 37°C for 30 minutes.
4. **Washing**: Wash 5 times with diluted wash buffer.
5. **Enzyme conjugate**: Add 50 μl enzyme reagent to each well.
6. **Second incubation**: Incubate for 30 minutes.
7. **Color development**: Add 50 μl each of TMB A and B, incubate at 37°C for 15 minutes.
8. **Stop reaction**: Add 50 μl stop solution.
9. **Reading**: Measure OD at 450 nm within 15 minutes.
**Important Notes**
- Allow the kit to equilibrate at room temperature before use.
- Avoid cross-contamination by using a new sealing film for each test.
- Handle all reagents carefully and follow the manual strictly.
- Always include a blank control and run standards in duplicate.
- If sample OD exceeds that of the highest standard, dilute the sample before testing.
- Maintain proper dilution factors during calculation.
**Calculation**
Plot the standard curve using concentration vs. OD values. Determine the sample concentration from the curve and multiply by the dilution factor. Alternatively, use linear regression analysis for more accurate results.
**Performance**
- Correlation coefficient (R²) ≥ 0.95
- Intra-assay CV < 9%, Inter-assay CV < 11%
- Detection range: 0.3–15 μg/L
**Storage and Shelf Life**
- Store the kit at 2–8°C.
- Validity: 6 months from the date of manufacture.
This guide provides a detailed overview of the Rat Histamine ELISA Kit, ensuring accurate and reliable results when used correctly. Always refer to the latest version of the manual for any updates or modifications.
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