I.Sample Processing and Requirements

1.Serum: Place whole blood specimens collected in serum separator tubes at room temperature for 2 hours or at 4°C overnight, then centrifuge at 1000×g for 20 minutes. Remove the supernatant and store at -20°C or -80°C. Avoid repeated freezing and thawing.

2.Plasma: Collect specimens using EDTA or heparin as an anticoagulant. Within 30 minutes of collection, centrifuge at 1000×g for 15 minutes at 2-8°C. Remove the supernatant and test. Alternatively, store at -20°C or -80°C. Avoid repeated freezing and thawing. 3. Tissue Homogenization: Rinse the tissue with pre-chilled PBS (0.01M, pH 7.4) to remove any residual blood (lysed red blood cells in the homogenate will affect the measurement results). Weigh the tissue and mince it. Add the minced tissue to the appropriate volume of PBS (generally a 1:9 weight-to-volume ratio, e.g., 1g of tissue sample to 9mL of PBS. The specific volume can be adjusted according to experimental needs and recorded. It is recommended to add protease inhibitors to the PBS) in a glass homogenizer and grind thoroughly on ice. To further lyse tissue cells, the homogenate can be sonicated or repeatedly freeze-thawed. Finally, centrifuge the homogenate at 5000×g for 5-10 minutes. Remove the supernatant for analysis.

4.Cell culture supernatant or other biological specimens: Centrifuge at 1000×g for 20 minutes. Remove the supernatant for testing, or store at -20°C or -80°C, but avoid repeated freezing and thawing.

Note: Hemolysis of the specimen will affect the final test results, so hemolyzed specimens are not suitable for this test.

II.Reagent Preparation

After removing the kit from the refrigerator, allow it to equilibrate to room temperature before use.

Dilution of 20×Wash Buffer: Dilute 1:20 with distilled water, i.e., add 1 part 20×Wash Buffer to 19 parts distilled water.

III.Procedure

1. After equilibration at room temperature for 20 minutes, remove the desired strips from the aluminum foil bag. Seal the remaining strips in a ziplock bag and return them to 4°C.

2. Set up standard wells and sample wells. Add 50 μL of standard solution of varying concentrations to each standard well.

3. Add 50 μL of the sample to be tested to the sample wells; leave blank wells untouched.

4. Add 100 μL of horseradish peroxidase (HRP)-labeled detection antibody to each standard well and sample well, except for the blank well. Seal the wells with plate sealing film and incubate at 37°C in a water bath or incubator for 60 minutes. 5. Discard the liquid, pat dry on absorbent paper, and fill each well with wash solution (350 μL). Let stand for 1 minute, then discard the wash solution and pat dry on absorbent paper. Repeat this process five times (a microplate washer can also be used). 6. Add 50 μL each of substrates A and B to each well and incubate at 37°C in the dark for 15 minutes. 7. Add 50 μL of stop solution to each well and measure the OD value of each well at 450 nm within 15 minutes.

Calculation of experimental results

Use the OD value of the measured standard as the horizontal axis and the concentration value of the standard as the vertical axis. Draw a standard curve on coordinate paper or using related software, and obtain a linear regression equation. Substitute the OD value of the sample into the equation to calculate the concentration of the sample.


Standard curve

Note: Repeatability: The intra-plate coefficient of variation is less than 10%, and the inter-plate coefficient of variation is less than 15%.

10mL

Remarks:

1. The concentrations of the standards are: 1000, 500, 250, 125, 62.5, and 31.25 pg/mL, respectively.
2. After testing a large number of normal specimens, the normal concentration values of the specimens are all within the detection range provided by the kit. During the experiment, 50 μL of sample can be directly taken for loading. When some sample values exceed the maximum standard concentration, the sample can be appropriately diluted with sample diluent before conducting the experiment.

1. Strictly follow the specified incubation time and temperature to ensure accurate results. All reagents must reach room temperature (20-25°C) before use. Refrigerate reagents immediately after use.

2. Improper plate washing can lead to inaccurate results. Ensure that the liquid in the wells is as dry as possible before adding substrate. Do not allow the microwells to dry out during incubation.

3. Remove any residual liquid and fingerprints from the bottom of the plate, otherwise it will affect the OD value.

4. The substrate developer solution should be colorless or very light in color. Substrate solution that has turned blue should not be used.

5. Avoid cross-contamination of reagents and specimens to prevent erroneous results.

6. Avoid direct exposure to strong light during storage and incubation.

7. Allow the sealed bag to equilibrate to room temperature before opening to prevent water droplets from condensing on the cold plate strips.

8. No reaction reagents should come into contact with bleaching solvents or the strong fumes emitted by bleaching solvents.

Any bleaching component will destroy the biological activity of the reagents in the kit. 9. Do not use expired products. 10. If there is a possibility of disease transmission, all samples should be managed properly and the samples and detection devices should be handled according to the prescribed procedures.