Research on Zebrafish Dopamine (DA) ELISA Detection Kit

Research on Zebrafish Dopamine (DA) ELISA Detection KitSummary of standard operating instructions for reference. Please refer to the actual reagent kit manual for specific steps.

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1、 Preparation before the experiment

1. Reagents and Materials

   • Reagent kit (including standard samples, detection antibodies, enzyme-linked immunosorbent assay (ELISA) plates, substrates A/B, stop buffer, washing solution, sealing membrane, etc.).
   • Samples (serum, plasma, tissue homogenate, etc.).
   • 37 ℃ constant temperature box, centrifuge, pipette, plate washer (or manual plate washer), enzyme-linked immunosorbent assay reader (450nm wavelength).

2. sample processing

   • Serum/plasma: Centrifuge (3000 × g, 10 minutes) and collect the supernatant.
   • Tissue homogenate: After homogenizing, centrifuge (5000 × g, 10 minutes) and take the supernatant.
   • The sample should avoid repeated freeze-thaw cycles and be stored at 4 ℃ for a short period of time or -80 ℃ for a long period of time.

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2、 Operation steps

1. Standard product preparation

• Dissolve the standard freeze-dried powder in diluent and dilute it in a gradient according to the instructions.

2. Sample addition

• Add 100 μ L of sample or standard to each well and set up blank wells.
• Cover with a sealing film and incubate at 37 ℃ for 90 minutes.

3. Washing

• Discard the liquid in the well, add 350 μ L of washing solution to each well, let it stand for 30 seconds, then discard, repeat 3 times. Shoot off any remaining liquid.

4. Detection of antibody incubation

• Add 100 μ L of biotinylated antibody working solution to each well and incubate at 37 ℃ for 60 minutes.
• Wash 3 times (same as step 3).

5. Color reaction

• Add 100 μ L enzyme conjugate (HRP labeled) to each well and incubate at 37 ℃ in the dark for 30 minutes.
• Wash 5 times (chedi removes washing solution).

6. Termination reaction and reading

• Add 50 μ L of termination solution to each well, and measure the OD value at 450nm wavelength using an enzyme-linked immunosorbent assay reader within 15 minutes.

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3、 Result calculation

1. Draw standard curve
   • Fit a standard curve (commonly used Logistic four parameter model) with standard concentration as the horizontal axis and OD value as the vertical axis.
2. Sample concentration calculation
   • Calculate the concentration of LPA by substituting the sample OD value into the standard curve formula.

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4、 Precautions

1. Strictly avoid cross contamination and replace the nozzle when adding samples.
2. The washing steps need to be timed to avoid residue affecting the signal.
3. Standard samples and reagents need to be restored to room temperature before use.
4. The termination liquid is corrosive and gloves should be worn during operation.
5. If the OD value exceeds the standard curve range, the sample dilution ratio needs to be adjusted.

Research on Zebrafish Dopamine (DA) ELISA Detection Kit
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