**Rat Histamine ELISA Technical Note**
This reagent is intended for research use only and not for human or animal diagnostic purposes. The kit is designed to quantitatively measure histamine levels in rat serum, plasma, urine, cell culture supernatants, and other biological fluids.
**Principle of the Assay**
The Rat Histamine ELISA Kit utilizes a double-antibody sandwich method. A microplate is pre-coated with a specific monoclonal antibody against rat histamine. After adding the sample, histamine binds to the immobilized antibody. An HRP-conjugated secondary antibody is then added, forming a complex of antibody–antigen–HRP-labeled antibody. Following washing steps, TMB substrate is introduced. Under the catalytic action of HRP, TMB turns blue and then yellow upon addition of the stop solution. The intensity of the color is directly proportional to the histamine concentration in the sample. Absorbance is measured at 450 nm using a microplate reader, and the histamine concentration is determined by comparing the OD values to a standard curve.
**Kit Components**
- Microplate (48-well or 96-well configuration)
- Standard: 18 μg/L, 0.5 mL × 1 bottle
- Standard Diluent: 1.5 mL × 1 bottle
- Enzyme-labeled Reagent: 3 mL × 1 bottle
- Sample Diluent: 3 mL × 1 bottle
- TMB Substrate A & B: 3 mL × 1 bottle each
- Wash Buffer (20×): 20 mL × 20 times or 20 mL × 30 times
- Sealing Film: 2 pieces per kit
- Storage: All components should be stored at 2–8°C.
**Sample Preparation Guidelines**
1. **Serum**: Allow blood to clot at room temperature for 10–20 minutes, centrifuge at 2000–3000 rpm for 20 minutes. Collect supernatant carefully; if precipitate forms, re-centrifuge.
2. **Plasma**: Use EDTA or sodium citrate as anticoagulant. Mix well, centrifuge, and collect supernatant.
3. **Urine**: Collect in sterile tubes, centrifuge, and collect supernatant.
4. **Cell Culture Supernatant**: Centrifuge at 2000–3000 rpm for 20 minutes. For intracellular components, lyse cells via freeze-thaw cycles, then centrifuge again.
5. **Tissue Homogenate**: Weigh tissue, homogenize in PBS (pH 7.4), centrifuge, and collect supernatant.
6. **Storage**: Samples should be processed as soon as possible. If not tested immediately, store at -20°C. Avoid repeated freeze-thaw cycles.
7. **Avoid NaN3**: Sodium azide inhibits HRP activity and must be excluded from samples.
**Procedure Summary**
1. Prepare standard dilutions (12 μg/L to 1 μg/L).
2. Add 40 μL sample diluent and 10 μL sample to each test well.
3. Incubate at 37°C for 30 minutes.
4. Wash 5 times with diluted wash buffer.
5. Add 50 μL enzyme-labeled reagent to each well (except blank).
6. Incubate again for 30 minutes.
7. Add 50 μL TMB A and B, incubate at 37°C for 15 minutes.
8. Stop reaction with 50 μL stop solution.
9. Measure OD at 450 nm within 15 minutes.
**Notes and Recommendations**
- Allow the kit to equilibrate at room temperature for 15–30 minutes before use.
- Do not reuse sealing films to prevent cross-contamination.
- Ensure accurate pipetting and calibration.
- Always prepare a standard curve and run duplicates.
- If sample OD exceeds that of the highest standard, perform a preliminary dilution.
- Keep all reagents away from light.
- Follow the manual strictly and rely on instrument readings for results.
- Treat all waste materials as biohazardous.
- Do not mix reagents from different batches.
**Calculation**
Plot the standard curve using concentrations vs. OD values. Calculate the unknown sample concentration using linear regression. Multiply by the dilution factor to obtain the actual value.
**Performance**
- Intra-assay and inter-assay CV < 9% and < 11%, respectively.
- Linear range: 0.3–15 μg/L.
- Correlation coefficient (R²) ≥ 0.95.
**Storage and Shelf Life**
- Store at 2–8°C.
- Shelf life: 6 months from date of manufacture.
This kit provides a reliable and sensitive method for histamine detection in rat samples, ideal for research applications.
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