Quantifying Plasma IL32 Levels

Blood samples were collected after an overnight fasting. For plasma collection, blood was centrifuged at 200× g for 15 min and immediately stored at −80 °C at the Fondazione Biological Resource Center (POLI-MI Biobank, which is part of the Italian node of Biobanking and Biomolecular Resources Research Infrastructure, BBMRI). Plasma samples were thawed only once and circulating levels of IL32 were quantified in duplicate using Human IL32 DuoSet ELISA kit (Cat. N° DY3040, R & D Systems, Minneapolis, MN, USA) following the manufacturer’s instructions. The assay is designed to detect IL32α, IL32β and IL32γ with a detection range of 78.5–5000 pg/mL. Samples were diluted 1:2–1:20 in PBS and were measured in duplicate. Analyses were repeated for further dilution or no dilution when values outside scale were detected. Optical density was measured at 450 nm using TECAN infinite F200 PRO instrument (Männedorf, Switzerland). The minimal detectable concentration above blank was 39 pg/mL. When the IL32 concentration was not detectable, it was assumed to be 10 pg/mL [20 (link)]. The intra-assay coefficient of variation (CV) was 3.9 ± 5.0%.