Multiplex magnetic bead based immunoassays

The plasma protein concentrations of ADAMTS13, eotaxin, Fas, GDF15, GROα, ICAM1, IL1α, IL6, IL7, IL8, IL10, IL15, MCP1, MDC, MIP1α, MIP1β, MMP1, MMP7, MMP9, MPO, OPN, PAI1, PARC, RAGE, RANTES, SOST, TARC, TNFR1, TNFR2, TNFα, and VEGFA were quantified using commercially available multiplex magnetic bead-based immunoassays (R&D Systems) on the Luminex xMAP multianalyte profiling platform and analyzed on MAGPIX System (Merck Millipore). Activin A concentration was determined by a Quantikine ELISA Kit (R&D Systems). Protein names, abbreviations, and aliases are provided in Additional file 1: Table S1. All assays were performed according to the manufacturer’s protocols. In instances where a biomarker was below the limit of detection in a participant sample, a value of half of the lowest measured value for that analyte was assigned.

The concentrations of protein biomarkers were quantified in plasma samples collected at baseline, 12‐ and 24‐month follow‐up timepoints using commercially available multiplex magnetic bead‐based immunoassays (R&D Systems) on the Luminex xMAP multianalyte profiling platform and analyzed on a MAGPIX System (Merck Millipore). All assays were performed according to the manufacturer's protocols. Twenty‐seven biomarkers were quantified with this method: ADAMTS13, eotaxin, Fas, GDF15, ICAM1, IL6, IL7, IL8, IL15, MDC, MMP1, MMP2, MMP7, MMP9, MPO, OPN, PAI1, PARC, RAGE, RANTES, SOST, STC1, TARC, TNFα, TNFR1, TNFR2, and VEGF. Activin A concentration was determined by a Quantikine ELISA Kit (R&D Systems) according to the manufacturer's instructions. In instances where a biomarker was below the limit of detection in a participant's sample, a value of half of the lowest measured value for that analyte was assigned. Assay performance characteristics are reported in Table S3.

Proteins were selected based on previous work (Schafer et al., 2020 (link)), and full names and abbreviations are shown in Supplemental Table 2. The concentrations of target proteins were quantified using commercially available multiplex magnetic bead‐based immunoassays (R&D Systems) on the Luminex xMAP multianalyte profiling platform and analyzed on MAGPIX System (Merck Millipore) using 220 μL of plasma. MMP9, MMP2, PARC, RANTES, PAI1 and MPO were analyzed on a standard 6‐plex panel using 20 μL plasma diluted 1:100. MMP1, ICAM1, GDF15, TNFRI, uPAR, and Fas were run on a standard 8‐plex panel using 30 μL plasma diluted 1:2. MMP7, MDC, RAGE, SOST, OPN, and TNFR2 were assessed as part of a standard 8‐plex panel using 30 μL plasma diluted 1:2. Eotaxin, MCP1, PDGF‐AA, PDGF‐AB, and VEGF were assessed as part of a performance 12‐plex assay using 30 μL plasma diluted 1:2. TNFα and IL7 were run on a high‐sensitivity 4‐plex panel using 55 μl plasma diluted 1:2 (100ul assay volume). Activin A concentration was determined by a Quantikine ELISA Kit (R&D Systems) using 55 μl plasma diluted 1:2. All assays were performed according to the manufacturer's protocols. Assay performance characteristics are reported in Supplemental Table 7, and a matrix showing the correlations between the SASP biomarkers is presented in Supplemental Table 8.