2.4 ELISA
Elisa kits for MMP-9 were purchased from R&D System Inc(Catalog No: DM-900). Serum samples were thawed under room temperature for 10 minutes, diluted at 1:100, and then transferred to polypropylene tubes. The wells added with 100ul of Assay Diluent were incubated for 2 hours at room temperature on a horizontal orbital microplate shaker with 100ul of standard or samples. After washing with Wash Buffer for 4 times, 200ul of Human MMP-9 Conjugate was added per well. Following one-hour incubation, the wells were washed and added with 200ul of Substrate Solution to react for 30 minutes. Ultimately, each well was added with 50ul of stop solution, and the optical density was determined with a microplate reader set to 450nm.