Human cardiac troponin 1

Enzyme-Linked Immunosorbent Assays (ELISA) were performed for the quantification of serum biomarkers. The manufacturer’s protocol was followed for each of the following ELISA Kit: Human IL-6 ELISA (Abcam), Human MMP2 ELISA (Abcam), Human MMP9 ELISA (Abcam), Human Myeloperoxidase (MPO) ELISA (Abcam), Human Topoisomerase II beta ELISA (MyBioSource). ELISAs were also performed for human cardiac troponin I (Abcam) and human cardiac Troponin T (MyBioSource). These assays were performed in a specific manufacturer provided antigen coated 96-well plate and at the end of protocol, the plate was read at 450 nm wavelength, in BioTek ELx900 Absorbance Reader^{12 (link)}. The standard curve was generated by plotting concentration of standards (x-axis) against corresponding absorbance (O.D value) of the standard (y-axis), as a XY-scatter plot to get 8-point curve. A linear trendline was added to the graph to achieve line of best fit with coefficient of determination (R² value) of ≥ 0.98. The concentrations for each biomarker in each sample was calculated using the resulting equation from the line of best fit, as describe previously^{12 (link),41 (link),42 (link)}.

Plasma samples were used for the quantitative assessment of biomarkers by enzyme linked immunosorbent assays (ELISAs). Commercially available kits were used and the manufacturer’s protocol was followed for each of the following biomarkers: Human GDF-15 (Abcam, United States), Human PIGF (Abcam, United States), Human cardiac MLC1 (MyBioSource, United States), Human cardiac Troponin T (MyBioSource, United States) and Human cardiac Troponin I (Abcam, United States). Each assay was performed using technical duplicates for each sample to minimize statistical error. The manufacturer’s provided antigen-specific coated 96-well plate was used for each assay, and the color produced at the end of the assay was read at 450nm wavelength in BioTek ELx900 Absorbance Reader, as described previously. The concentrations for each biomarker in each sample was calculated based on the standard curve, and the resulting equation from the line of best fit.