1. Sample processing and requirements

1. Serum: Place the whole blood sample collected in the serum separation tube at room temperature for 2 hours or 4 ℃ overnight, then centrifuge at 1000 × g for 20 minutes, take the supernatant, or store the supernatant at-20 ℃ or-80 ℃, but avoid repeated freezing and thawing.
2. Plasma: Use EDTA or heparin as anticoagulant to collect specimens, and centrifuge the specimens at 2-8 ℃ 1000 × g for 15 minutes within 30 minutes after collection. Take the supernatant for detection, or place the supernatant at-20 ℃ or-80 ℃ for storage, but avoid repeated freezing and thawing.
3. Tissue homogenization: with pre-cooled PBS（0.01 M, pH = 7.4）The tissue is flushed to remove residual blood (lysed red blood cells in the homogenate can affect the measurement), and the tissue is weighed and cut into pieces. Combine the chopped tissue with the corresponding volume of PBS (generally according to the weight-to-volume ratio of 1: 9, for example, 1g of tissue sample corresponds to 9mL of PBS. The specific volume can be appropriately adjusted according to the needs of the experiment and recorded. It is recommended to add protease inhibitor to PBS) add to a glass homogenizer and grind thoroughly on ice. For further lysis of tissue cells, the homogenate can be sonicated, or freeze-thawed repeatedly. Finally, the homogenate was centrifuged at 5000 × g for 5-10 minutes, and the supernatant was taken for detection.
4. Cell culture supernatant or other biological specimens: Please centrifuge at 1000 × g for 20 minutes, take the supernatant for detection, or store the supernatant at-20 ℃ or-80 ℃, but avoid repeated freezing and thawing.

Note: Hemolysis of the specimen will affect the final test result, so hemolyzed specimens should not be tested.

2. Operation steps

1. The required slats were removed from the aluminum foil bag after equilibration at room temperature for 20 minutes, and the remaining slats were sealed with a ziplock bag and returned to 4 °C.
2. Set up standard wells and sample wells, and add 50μL of different concentrations of standards to each standard well;
3. Add 50μL of the sample to be tested to the sample well; Blank holes are not added.
4. In addition to the blank wells, 100 μL of horseradish peroxidase (HRP)-labeled detection antibody was added to each well of the standard wells and sample wells, the reaction wells were sealed with a plate-sealing membrane, and incubated for 60 minutes in a 37 ℃ water bath or incubator.
5. Discard the liquid, pat dry on absorbent paper, fill each well with washing liquid (350μL), let it stand for 1min, throw away the washing liquid, pat dry on absorbent paper, and repeat washing the plate 5 times (you can also use a plate washer to wash the plate).
6. Add 50 μL of each substrate A and B to each well, and incubate at 37 °C in the dark for 15 minutes.
7. 50 μL of stop solution was added to each well, and the OD value of each well was measured at a wavelength of 450 nm within 15 minutes.

Calculation of experimental results

Taking the OD value of the measured standard product as the abscissa and the concentration value of the standard product as the ordinate, draw the standard curve on coordinate paper or with relevant software, and get the linear regression equation. Substitute the OD value of the sample into the equation to calculate the concentration of the sample.

1. Detection range : 0.75 ng/mL-24 ng/mL.
2. Sensitivity: The lowest detection concentration is less than 0.1 ng/mL.
3. Specificity: Does not cross-react with other soluble structural analogs.
4. Repeatability: Intra-plate coefficient of variation less than 10% and inter-plate coefficient of variation less than 15%.

1. The standard concentrations were 24, 12, 6, 3, 1.5, 0.75 ng/mL in order
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, 50μL samples can be directly taken and loaded. When some sample values exceed the maximum standard concentration, the sample can be appropriately diluted with sample diluent before conducting the experiment.

1. Incubation was performed strictly according to the specified time and temperature to ensure accurate results. All reagents must reach room temperature 20-25 °C prior to use. Store reagents in refrigeration immediately after use.
2. Incorrect plate washing can lead to inaccurate results. Make sure to drain the liquid from the wells as much as possible before adding the substrate. Do not allow the wells to dry out during incubation.
3. Eliminate residual liquid and fingerprints on the bottom of the plate, otherwise affecting the OD value.
4. The color development solution of the substrate should be colorless or very light in color, and the substrate solution that has turned blue cannot be used.
5. Avoid cross-contamination of reagents and specimens to avoid wrong results.
6. Avoid direct exposure to bright light during storage and incubation.
7. After equilibrating to room temperature, open the sealed bag to prevent water droplets from agglomerating on the cold slats.
8. Any reagent must not come into contact with the bleaching solvent or the strong gases emitted by the bleaching solvent. Any bleaching component will destroy the biological activity of the reaction reagents in the kit.
9. Expired products cannot be used.
10. If it is possible to spread disease, all samples should be managed, and the samples and testing devices should be handled according to the prescribed procedures.