Quantification of Human MMP-9 by ELISA

The test was performed with the Human MMP-9 ELISA kit from Invitrogen, BenderMedSystems GmbH, Vienna, Austria, as described earlier [52 (link)]. Briefly, the 96-well assay plate coated with anti-MMP-9 antibody was washed and then loaded with 100 µL standards (9 serial concentrations in the range of 0.017–7500.00 ng/mL, in duplicates), 100 µL of assay buffer as blank, 100 µL of samples, in duplicates. A total of 50 µL biotin-conjugated anti-MMP-9 antibody solution was dispensed in each well, and the plate was incubated with shaking for 2 h. The plate was washed four times, 100 µL of Streptavidin-HRP solution was added to each well, and the plate was incubated for 1 h. After another wash cycle, the wells were loaded with 100 µL TMB substrate and after 15 min, a stop solution was added to the plate, and the samples were read with the ELISA reader, at 540/450 nm. The standard curve was created, and individual concentrations were plotted by the Magellan software of the ELISA Equipment.