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The Fish Norepinephrine (NE) ELISA Kit is a double-antibody sandwich immunoassay designed for the quantitative determination of norepinephrine in fish serum, plasma, and other biological fluids. This kit is intended for research purposes only and should not be used for diagnostic or clinical applications.
**Principle of Operation:**
The assay is based on the competitive binding principle. A microplate is pre-coated with a specific antibody against fish norepinephrine. The sample or standard is added to the wells, followed by an HRP-labeled secondary antibody. This forms a complex of antibody-antigen-enzyme-labeled antibody. After washing, the substrate TMB is added, which changes color in the presence of HRP. The intensity of the color is directly proportional to the concentration of norepinephrine in the sample. The reaction is stopped with a stop solution, and the absorbance is measured at 450 nm using a microplate reader. A standard curve is then generated, and the sample concentration is calculated accordingly.
**Kit Components:**
The kit includes all necessary reagents for 48 or 96 tests, depending on the configuration. It contains:
- Microtiter plates coated with anti-NE antibodies
- Standard solutions (ranging from 3 ng/L to 120 ng/L)
- Enzyme-labeled antibody
- Dilution buffers
- Substrate solutions (TMB A and B)
- Wash buffer (concentrated)
- Stop solution
- Sealing film and storage bags
**Storage Conditions:**
All components should be stored at 2–8°C. The shelf life is 6 months when stored properly. Before use, the kit should be allowed to equilibrate to room temperature for 15–30 minutes.
**Sample Preparation:**
Proper sample handling is crucial for accurate results. For serum and plasma, samples should be centrifuged after collection. Urine and cell culture supernatants should also be centrifuged to remove particulates. Tissue samples must be homogenized in PBS before centrifugation. Avoid repeated freeze-thaw cycles and store samples at -20°C if not tested immediately.
**Procedure Overview:**
1. Prepare standard dilutions and add them to designated wells.
2. Add sample diluent and test samples to the appropriate wells.
3. Seal the plate and incubate at 37°C for 30 minutes.
4. Wash the plate five times with diluted wash buffer.
5. Add the enzyme-labeled reagent and incubate again.
6. Add TMB substrate and incubate for 15 minutes in the dark.
7. Stop the reaction with the stop solution.
8. Measure OD values at 450 nm within 15 minutes.
9. Calculate concentrations using the standard curve.
**Notes and Precautions:**
- Ensure all reagents are at room temperature before use.
- Do not mix reagents from different batches.
- Avoid exposure of the substrate to light.
- Always run a blank control and duplicate samples for accuracy.
- If sample OD exceeds the highest standard, perform a preliminary dilution.
- Discard all waste as biohazardous material.
**Performance Characteristics:**
- The correlation coefficient (R²) of the standard curve is ≥0.95.
- Intra-assay and inter-assay variability are <9% and <11%, respectively.
- Detection range: 3 ng/L – 120 ng/L.
This kit provides a reliable and sensitive method for measuring norepinephrine levels in fish samples, making it a valuable tool in physiological and environmental studies.