2.5. Detection Methods

Four mL of fasting venous blood was extracted an hour before scanning, and serum was extracted by coagulation, centrifuged at 1,200 rpm for 10 min and stored at −80 °C. After the collection of all patients, the previously frozen serum was thawed and mixed by shaking and then diluted with FBS buffer at a dilution ratio of 1 : 350. Antibody cross-linking microspheres were prepared after dilution, and standard lines of quality control, lotion, serum matrix, and cytokine were prepared. In the ELISA test, 150 μL reaction buffer was added to each well of the reaction plate, shaken at room temperature for 15 min, which was then filtered or discarded with absorbent paper. Then, 20 μL reaction buffer was added to the sample wells, and 20 μL standard and quality control were added to the corresponding wells; 20 μL matrix solution was added to standard wells, quality control, and background, and 20 μL samples were added to the sample well, oscillated at room temperature for 15 min. Then, 20 μL mixed microspheres were added to each well. The plate was sealed and reacted at room temperature for two hours, then filtered, washed with 150 μL PBS buffer solution twice, and dried with blotting paper. At room temperature, 20 μL antibody was added to each well, and then 25 μL Streptavicin-Rhycoerythrin was added to each well. After suction filtration, the plate was washed with 50 μL PBS buffer solution twice, added with 150 μL sheathing solution, oscillated for 10 min, and then read.