The Human FETU-B ELISA Kit is designed for easy and accurate detection of FETU-B levels in biological samples. It does not require any additional reagents or equipment beyond what is provided in the kit. The following items are needed:
1. A microplate reader set at 450 nm
2. A high-precision pipette with tips ranging from 0.5–10 µL, 2–20 µL, 20–200 µL, and 200–1000 µL
3. A 37°C incubator
4. Distilled or deionized water
**Notes:**
After testing a large number of normal specimens, it was found that the normal concentration of samples typically falls within the detection range of the kit. Therefore, during the experiment, you can directly use 50 µL of the sample. However, if some sample values exceed the maximum standard concentration, it is recommended to dilute the sample with the provided sample diluent before proceeding with the test.
**Precautions:**
1. Incubation must strictly follow the specified time and temperature to ensure accurate results. All reagents should be brought to room temperature (20–25°C) before use and stored refrigerated immediately after use.
2. Inadequate washing may lead to inaccurate results. Make sure to remove as much liquid as possible from each well before adding the substrate. Keep the microplate moist during incubation.
3. Remove any residual liquid or fingerprints from the bottom of the plate, as they may interfere with the OD reading.
4. The substrate solution should be colorless or very light. If it turns blue, do not use it.
5. Avoid cross-contamination between reagents and samples to prevent errors.
6. Keep the kit away from direct strong light during storage and incubation.
7. After equilibrating to room temperature, open the sealed bag carefully to avoid condensation on the plate.
8. Do not expose any reagents to strong gases from bleaching agents or bleach, as these can damage the biological activity of the reagents.
9. Never use expired reagents.
10. If there is a risk of disease transmission, handle all samples according to established safety protocols and dispose of samples and test materials properly.
**Reagent Preparation:**
The 20× Wash Buffer should be diluted with distilled water at a ratio of 1:20 (i.e., 1 part of 20× wash buffer + 19 parts of distilled water).
**Procedure:**
1. After equilibrating the plate at room temperature for 20 minutes, remove the required strips from the foil pouch. Seal the remaining strips back in the ziplock bag and store them at 4°C.
2. Set up the standard and sample wells. Add 50 µL of each standard solution to the corresponding wells.
3. Add 50 µL of the 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 wells), cover with a sealing membrane, 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 it stand for 1 minute, then discard the buffer. Repeat this washing process 5 times (or use an automated washer if available).
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 value at 450 nm within 15 minutes.
**Data Analysis:**
Use the OD values of the standards as the x-axis and their concentrations as the y-axis to create a standard curve. Plot the curve on graph paper or using software, obtain the linear regression equation, and substitute the sample’s OD value into the equation to calculate its concentration.
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