Biomarker determinations in NDEV lysates
REGRESSION, EMD
We used enzyme-linked immunosorbent assays (ELISAs) to quantify the concentration of proBDNF (cat. no. BEK-2237; Biosensis) and humanin, also known as human putative humanin peptide or MT-RNR2 (cat. no. CSB-EL015084HU; Cusabio), in NDEV lysates; plates were read using the Synergy™ H1 microplate reader set to 450 nm and the Gen5™ microplate data collection software (BioTek Instruments). NDEV levels of BDNF, irisin, apelin, fractalkine, erythropoeitin (EPO), osteonectin (SPARC), interleukin-15 (IL-15), myostatin (MSTN)/GDF8, FABP3, follistatin-like 1 protein (FSTL-1), oncostatin-M (OSM) and osteocrin/musclin were quantified utilizing a Milliplex® Human Myokine Magnetic Bead panel (cat. no. HMYOMAG-56 K; EMD Millipore Corporation). Milliplex plates were read using the Luminex® 100/200™ instrument with the xPOTENT® acquisition software (Luminex Corporation). NDEV and panTET-EV samples were run undiluted based on experiments determining the optimal input volume for each assay. Standard curve equations of ELISAs were determined using four-parameter logistic (4-PL) regression, whereas for Milliplex assays, protein concentrations were extrapolated from a five-parameter logistic (5-PL) curve.Samples were assessed in duplicate in all assays. The limit of detection (LOD) was defined as mean signal of the blank plus 2.5 times its standard deviation (SD). The lowest limit of quantification (LLOQ) was set by the following rules: (1) signal above LOD, (2) coefficient of variation (CV) > 20%, and (3) 80–120% recovery. A zero value was imputed to samples with signals below LOD; the LLOQ was imputed for signals between LOD and LLOQ; duplicate measurements with %CV ≥ 30 were excluded from the analysis. Excluded samples per analyte: 2 for proBDNF, 15 for humanin in NDEVs, 0 for humanin in panTET-EVs, 27 for apelin, 10 for fractalkine, 2 for BDNF, 2 for EPO, 8 for SPARC, 1 for IL-15, 3 for MSTN/GDF8, 13 for FABP3, 30 for irisin, 28 for FSTL-1, 21 for OSM and 21 for osteocrin/musclin.An internal control (IC) was included in all plates to assess inter-plate variability. NDEVs from a healthy participant and a quality control provided were used as ICs for ELISAs and the Milliplex assay, respectively. CVs for the ICs across all plates were below 30% and hence, signals were not normalized (CVs for proBDNF, 9.9%; humanin, 23.1%; apelin, 5.3%; fractalkine, 11.8%; BDNF, 21.7%; EPO, 3.8%; SPARC, 8.4%; IL-15, 8.7%; MSTN/GDF8, 9.8%; FABP3, 12.0%; irisin, 3.6%; FSTL-1, 9.3%; OSM, 7.4%; and osteocrin/musclin, 17.3%).