Quantitative elisa

Plasma levels of syndecan-1 (Syn1) and hyaluronic acid (HA) were performed using quantitative ELISA (Thermo Scientific, MD, USA) following manufacturer's instructions at admission for the discovery cohort, and at admission and 60 days post-discharge for the validation cohort. Magnetic bead-based multiplex assay performed in a Luminex® platform (R&D Systems, MN, USA) were used to quantify plasma concentration of lumican, matrix metalloproteinase (MMP)2, tissue inhibitors of MMP (TIMP)1, TIMP2. These proteins were assayed at admission for the discovery cohort, and at admission and 60 days post-discharge for the validation cohort. Further, cytokine and chemokines (n=29) concentration in plasma at admission and 60 days post-discharge for the validation cohort were determined by using a human cytokine magnetic bead assay (EMD Millipore) on the Magpix with Xponent software (version 4.2; Luminex Corp) and acquired Median Fluorescent Intensity data analysed using the Milliplex analyst software (version 3.5.5.0 standard). Levels of CXCL-13, MMP3, 8, 13, and TGF-β were also measured but their concentrations were too low to be detected in most of the samples.

Levels of these substances were assessed using immunoassay array (Human Cytokine Antibody Array 5, RayBio, Norcross, GA, USA) and quantified by ImageJ software program (https://imagej.nih.gov/ij/). Levels of individual cytokines were determined by quantitative ELISA (Thermo Fisher).