Plasma total MMP-3 Protein Measurement using ELISA
WELLS
All plasma samples were stored at − 80 °C until use. Plasma MMP-3 concentrations were measured with Human Total MMP-3 DuoSet ELISA Kit from R&D Systems, Inc, Catalog #: DY513 (Minneapolis, MN). Briefly, 100 μL of the sample (or control standard) and Reagent Diluent were added to each well. The plate was covered with an adhesive strip and incubated for 2 h at room temperature. Wells were aspirated and washed with Wash Buffer, repeating the wash process two times for a total of three washes. A 100 μL of the detection antibody in reagent diluent was added to each well. The plate was covered with a new adhesive strip and incubated for 2 h at room temperature. The aspiration and wash process was repeated three times. Then, 100 μL of the working dilution of Streptavidin-HRP was added to each well, and the plate was covered and incubated for 20 min at room temperature, followed by repeat aspiration and wash cycles. Following aspiration and wash, a 100 μL of substrate solution was added to each well and incubated for 20 min at room temperature. Lastly, add 50 μL of stop-solution (2N sulfuric acid) to each well. Optical density was determined at 450 nm. MMP-3 concentration was calculated based on a linear standard curve.