Sample handling and requirements

1.Serum:The whole blood sample collected in the serum separation tube is placed at room temperature for 2 hours or 4 ℃ overnight, and then centrifuged at 1000 × g for 20 minutes. The supernatant can be taken, or the supernatant can be stored at-20 ℃ or-80 ℃, but repeated freezing and thawing should be avoided.

2.Plasma:Specimens were collected using either EDTA or heparin as anticoagulant and subjected to the specimens within 30 minutes after collection at 2-8 °C 1000

× g centrifuge for 15 minutes, take the supernatant for detection, or store the supernatant at-20 ℃ or-80 ℃, but avoid repeated freezing and thawing.

3.Tissue homogenate:The tissues were washed with pre-cooled PBS (0.01 M, pH = 7.4) to remove residual blood (lysed red blood cells in the homogenate would affect the measurement results), and the tissues were sheared after weighing. 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 specimen: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.

Reagent Preparation

The kit should be removed from the refrigerated environment and equilibrated at room temperature before use.

Dilution of 20 × wash buffer: Distilled water was diluted 1:20, i.e. 1 part of 20 × wash buffer plus 19 parts of distilled water.

Operation steps

1. Take out the required slats from the aluminum foil bag after equilibration at room temperature for 20 minutes, and seal the remaining slats with a ziplock bag and put them back 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. Add horseradish peroxidase (HRP)-labeled detection antibody to each well of the standard well and sample well except for the blank well

100 μL, seal the reaction wells with a plate sealing membrane, and incubate for 60 minutes in a 37 °C 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 washing machine 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. Add 50 μL of stop solution to each well, and measure the OD value of each well at a wavelength of 450nm 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.

Standard curve

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. Incubate in strict accordance with 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 the residual liquid and fingerprints at the bottom of the plate, otherwise it will affect the OD value.

4. The substrate color development solution 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 strong light during storage and incubation.

7. After equilibrating to room temperature, open the sealed bag to prevent water droplets from condensing on the cold slats.

8. Any reaction reagent cannot come into contact with the bleaching solvent or the strong gas 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 diseases, all samples should be managed, and the samples and testing devices should be handled according to the prescribed procedures.