2.2.1. Determination of MMP-2

To measure MMP-2 concentrations, an ELISA kit from R&D Systems (Cat. No. DMP2F0) (Minneapolis, MN, USA) was used. According to the manufacturer's instructions, 100 μL of assay diluent RD1-74 was added to each well-plate, then 50 μL tested sera, diluted 1:10 with calibrator diluent RD5-32 (20 μL serum + 180 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 of the conjugate was added to each well and incubated for 2 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).
