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. Remove the desired strips from the aluminum foil bag after equilibration at room temperature for 20 minutes. 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 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 and sample well, except for the blank well. Seal the wells with sealing film and incubate at 37°C in a waterbath or incubator for 60 minutes. 5. Discard the liquid, pat dry on absorbent paper, and fill each well with wash buffer (350 μL). Let stand for 1 minute, then discard the wash buffer and pat dry on absorbent paper. Repeat this process five times (a plate 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. Within 15 minutes, measure the OD value of each well at a wavelength of 450 nm. 4. Calculation of Experimental Results Using the OD value of the measured standard as the horizontal axis and the concentration of the standard as the vertical axis, draw a standard curve on graph paper or using relevant software. Obtain a linear regression equation. Substitute the OD value of the sample into the equation to calculate the sample concentration.

Note: For reference only

Remarks:

1. The concentrations of the standards are: 16, 8, 4, 2, 1, and 0.5 ng/mL.

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 sampled. If some sample values exceed the maximum standard concentration, the specimens can be appropriately diluted with sample diluent before the experiment.

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.