**Human Ectodermal Dysplasia Protein 1 (EDA) ELISA Kit – Instructions and Precautions**
The Human EDA ELISA kit is designed for the quantitative detection of EDA in biological samples. The following reagents and equipment are required but not provided with the kit:
1. Microplate reader (wavelength: 450 nm)
2. Precision pipettes with tips: 0.5–10 μL, 2–20 μL, 20–200 μL, 200–1000 μL
3. Incubator set at 37°C
4. Distilled or deionized water
**Notes:**
After testing a large number of normal specimens, it was observed that most sample concentrations fall within the detection range of the kit. In such cases, 50 μL of the sample can be directly used. However, if some samples exceed the maximum standard concentration, it is recommended to dilute them using the provided sample diluent before proceeding with the experiment.
**Precautions:**
1. Strictly follow the incubation time and temperature specified in the protocol to ensure accurate results. All reagents should be brought to room temperature (20–25°C) before use and stored properly after each use.
2. Proper washing is critical to avoid false readings. Ensure all liquid is removed from the wells before adding the substrate. Keep the microplate moist during incubation.
3. Remove any residual liquid or fingerprints from the bottom of the plate to prevent interference with OD readings.
4. The substrate solution should be colorless or very pale. If it turns blue, discard it immediately.
5. Prevent cross-contamination between reagents and samples to avoid inaccurate results.
6. Avoid exposing the kit components to strong light during storage or incubation.
7. After equilibrating to room temperature, open the sealed pouch carefully to prevent condensation on the plate.
8. Avoid contact with bleach or other volatile substances, as they may degrade the reagents.
9. Do not use expired kits or reagents.
10. Handle all samples as potential biohazards and follow appropriate safety protocols.
**Reagent Preparation:**
Dilute the 20× Wash Buffer by mixing 1 part of the buffer with 19 parts of distilled water.
**Procedure:**
1. Allow the microplate to equilibrate at room temperature for 20 minutes. Remove the required strips from the foil pouch and return the unused strips to the refrigerator in a sealed bag.
2. Set up the standard and sample wells. Add 50 μL of standard solutions to each standard well.
3. Add 50 μL of the sample to be tested in the corresponding sample wells. Leave the blank wells empty.
4. Add 100 μL of HRP-conjugated detection antibody to each well (except blank wells), cover with a sealing film, and incubate at 37°C for 60 minutes.
5. Discard the liquid, blot dry with absorbent paper, and wash each well with 350 μL of wash buffer. Let stand for 1 minute, then remove the buffer. Repeat this process 5 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. Add 50 μL of stop solution to each well, and measure the OD at 450 nm within 15 minutes.
**Data Analysis:**
Plot the OD values of the standards against their known concentrations to create a standard curve. Use linear regression to determine the equation of the curve. Input the OD value of the unknown sample into the equation to calculate its EDA concentration.
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