Duosets

Mouse and human IL-1β DuoSets were obtained from R&D Systems (Minneapolis, MN) and ELISA assays performed according to the manufacturer’s protocol. IL-18 capture and detection antibodies were also obtained from R&D Systems. The IL-18 ELISA, although developed in-house, was run similar to R&D Systems IL-33 DuoSet ELISA with regard to timings, diluents, standard curves, and washes. Lavage fluid samples were assayed without dilution. In vitro media supernatants were diluted (1:5 for mouse cells, 1:100 for human cells) for optimizing the fit to the kit’s standard curve. All plates were read at 450 nm and data expressed as pg/ml.
Cathepsin activity for the first WLL fluid was determined by mixing the following assay components in a 96-well plate using PBS as diluent: first WLL fluid (50 μl), 2 μg Z-LR-AMC (fluorogenic Peptide Substrate, R&D systems, Minneapolis, MN) in a total volume of 150 μl. The assays samples were incubated at 37 °C for 1 hour then fluorescence was measured using a plate reader at 380 nm excitation and 460 nm emission.

Maternal peripheral blood samples were collected in EDTA vacutainer tubes at enrolment, plasma separated, and stored at −80°C prior to testing. For the present study, plasma samples underwent analysis for the following 18 angiogenic and inflammatory biomarkers: angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2), angiopoietin-like 3 (Angptl3), vascular endothelial growth factor (VEGF-A), soluble fms-like tyrosine kinase 1 (sFlt-1), soluble tumor necrosis factor receptor 2 (sTNFR2), placental growth factor (PGF), macrophage inflammatory protein-1 beta (MIPβ/CCL4), monocyte chemoattractant protein-1 (MCP-1/CCL2), Leptin, interleukin-1 beta (IL1β), interleukin-18 binding protein (IL-18BP), soluble intercellular adhesion molecule-1 (sICAM1), Complement Factor D (Factor D), soluble endoglin (sEng), C-reactive protein (CRP), chitinase-3-like protein-1 (CHI3L1), and complement component C5a (C5a). All analyses utilized commercially available ELISAs (Duosets, R&D Systems, Minneapolis, MN). To increase sensitivity, samples were incubated for two hours at room temperature (18-28°C) for the analyses of CRP, C5a, Factor D and VEGF-A, and overnight at 4°C for the analyses of all other biomarkers. ELISA analysis was blinded to infant size for gestational age at birth.