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**Fish Norepinephrine (NE) ELISA Double-Antibody Sandwich Method**
This reagent is intended for research purposes only and is designed to quantify norepinephrine (NE) in fish serum, plasma, and related biological fluids. The method employed is a double-antibody sandwich ELISA, ensuring high specificity and accuracy.
**Principle of the Assay**
The assay utilizes a microplate pre-coated with a specific antibody against fish norepinephrine. After adding the sample, NE binds to the immobilized antibody. A horseradish peroxidase (HRP)-labeled secondary antibody then recognizes the captured NE, forming a complex. Following incubation and washing, the substrate TMB is added. HRP catalyzes the conversion of TMB into a blue color, which turns yellow upon addition of an acidic stop solution. The intensity of the color correlates directly with the concentration of NE in the sample. The absorbance is measured at 450 nm using a microplate reader, and the NE concentration is determined by comparing the sample OD values to a standard curve.
**Kit Components**
- 48-well or 96-well configuration
- Microtiter plate coated with anti-NE antibody
- Standard solutions (135 ng/L, 0.5 mL × 1 bottle)
- Standard diluent (1.5 mL × 1 bottle)
- Enzyme-labeled antibody (3 mL × 1 bottle)
- Developer A and B (3 mL × 1 bottle each)
- Wash buffer (20× concentrated, 20 mL × 20 times or 30 times)
- Sample diluent (3 mL × 1 bottle)
**Sample Preparation**
- **Serum:** Allow blood to clot at room temperature for 10–20 minutes, then centrifuge at 2000–3000 rpm for 20 minutes. Collect supernatant.
- **Plasma:** Use EDTA or sodium citrate as anticoagulant, mix for 10–20 minutes, then centrifuge.
- **Urine:** Centrifuge at 2000–3000 rpm for 20 minutes.
- **Cell culture supernatant:** Centrifuge after collection; for intracellular components, lyse cells via freeze-thaw cycles before centrifuging.
- **Tissue:** Homogenize in PBS (pH 7.4), centrifuge, and collect supernatant.
**Operation Steps**
1. Prepare standards by serial dilution (90 ng/L, 60 ng/L, 30 ng/L, 15 ng/L, 7.5 ng/L).
2. Add 40 µL sample diluent and 10 µL sample to each test well.
3. Seal and incubate at 37°C for 30 minutes.
4. Wash 5 times with diluted wash buffer.
5. Add 50 µL enzyme-labeled antibody to each well.
6. Incubate again at 37°C for 30 minutes.
7. Add 50 µL each of developer A and B, incubate for 15 minutes at 37°C.
8. Stop reaction with 50 µL stop solution.
9. Measure OD at 450 nm within 15 minutes.
**Notes**
- Equilibrate kit components to room temperature before use.
- Avoid repeated freezing and thawing of samples.
- Do not use samples containing NaN3, as it inhibits HRP activity.
- Always prepare a standard curve and use duplicate wells for accuracy.
- Keep all reagents away from light and store at 2–8°C.
- Discard any unused portions of the sealing film to prevent contamination.
**Calculation**
Plot the standard curve using concentrations vs. OD values. Determine the sample concentration by interpolating its OD value on the curve and multiplying by the dilution factor. Alternatively, use linear regression for more precise results.
**Performance**
- Linear correlation coefficient (R²) ≥ 0.95
- Intra-batch and inter-batch variation < 9% and < 11%, respectively
- Detection range: 3 ng/L – 120 ng/L
**Storage & Expiry**
- Store kit at 2–8°C.
- Shelf life: 6 months from the date of manufacture.
This detailed procedure ensures accurate and reliable quantification of norepinephrine in various fish biological samples, making it ideal for research applications in aquatic physiology and stress response studies.