**Human Ectodermal Dysplasia Protein 1 (EDA) ELISA Kit: Procedures, Reagents, and Precautions**
The Human Ectodermal Dysplasia Protein 1 (EDA) ELISA kit requires certain reagents and equipment that are not included in the kit. Below is a detailed list of the necessary items:
1. **Microplate Reader**: A spectrophotometer capable of measuring absorbance at 450 nm.
2. **Precision Pipettes and Tips**: Ensure accuracy with a range of 0.5–10 µL, 2–20 µL, 20–200 µL, and 200–1000 µL.
3. **Incubator**: Set to 37°C for proper incubation during the assay.
4. **Distilled or Deionized Water**: Used for diluting reagents and washing steps.
**Notes**:
After testing a large number of normal specimens, it was observed that the typical concentration of samples falls within the detection range provided by the kit. Therefore, 50 µL of each sample can be directly used. However, if some sample values exceed the maximum standard concentration, the sample should be diluted using the provided sample diluent before proceeding with the test.
**Important Precautions**:
1. **Strict Incubation Conditions**: Follow the specified time and temperature precisely to ensure accurate results. All reagents must reach room temperature (20–25°C) before use, and they should be stored refrigerated immediately after use.
2. **Proper Washing**: Inadequate washing may lead to false results. Make sure to remove as much liquid as possible from each well before adding the substrate. Keep the microplates moist during incubation.
3. **Clean Plate Surfaces**: Remove any residual liquid or fingerprints from the bottom of the plate to avoid affecting OD readings.
4. **Substrate Quality**: The substrate solution should be colorless or very light. If it turns blue, it should not be used.
5. **Avoid Contamination**: Prevent cross-contamination between reagents and samples to ensure reliable outcomes.
6. **Light Protection**: Avoid exposing the kit or samples to strong light during storage or incubation.
7. **Equilibration Before Use**: After bringing the kit to room temperature, open the sealed bag carefully to prevent condensation on the plate wells.
8. **Reagent Stability**: Do not expose reagents to strong gases from bleach or other solvents, as this can degrade their biological activity.
9. **Use Before Expiration**: Never use expired reagents or kits.
10. **Biohazard Management**: If the sample is suspected to be infectious, handle all materials according to biosafety protocols.
**Reagent Preparation**:
- **Dilution of 20× Wash Buffer**: Mix 1 part of 20× wash buffer with 19 parts of distilled water to prepare the working solution.
**Procedure**:
1. Allow the microplate strips to equilibrate at room temperature for 20 minutes. Remove the required number of wells from the foil pouch and reseal the remaining ones in a ziplock bag at 4°C.
2. Label the standard and sample wells. Add 50 µL of each standard solution to the respective wells.
3. Add 50 µL of the test sample to the sample wells. Do not add anything to the blank wells.
4. Add 100 µL of HRP-conjugated detection antibody to each well (except the blank), seal with an adhesive film, and incubate at 37°C for 60 minutes.
5. Discard the liquid, blot dry on absorbent paper, and add 350 µL of wash buffer to each well. Let stand for 1 minute, then discard and repeat the washing process five times (or use an automated washer).
6. Add 50 µL of substrate A and B to each well, and incubate in the dark at 37°C for 15 minutes.
7. Stop the reaction by adding 50 µL of stop solution to each well. Measure the OD value at 450 nm within 15 minutes.
**Data Analysis**:
Plot the OD values of the standards against their concentrations on graph paper or using software. Determine the linear regression equation and use it to calculate the EDA concentration in the sample based on its OD value.
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