2.2.2. Determination of MMP-9

To measure MMP-9 concentrations, an ELISA kit from R&D Systems (Cat. No. DMP900) (Minneapolis, MN, USA) was used. According to the manufacturer's instructions, to each well-plate, 100 μL of assay diluent RD1-34 was added, then 100 μL tested sera, diluted 1:100 with calibrator diluent RD5-10 (10 μL serum + 990 μL calibrator diluent) or standards, was added at various concentrations to construct a calibration curve. After 2 h downtime at room temperature on a shaker, plates were washed three times with 400 μL wash buffer per well. After the last wash, 200 μL anti-MMP-9 antibody conjugated with peroxidase was added to each well and was incubated for 1 h at room temperature on a shaker. The plate was washed again three times, and in each well, 200 μL substrate solution was added. This was incubated for 30 min at room temperature in the dark. The reaction was stopped by adding 50 μL of stop solution to each well. Within 30 min, the serum samples were assayed at 450 nm on an automatic micro-ELISA plate reader (Coulter Microplate Reader UV Max).
