Ifabp

Levels of Endotoxin-core antibodies IgM (Hycult Biotech, Uden, Netherlands), soluble CD14 (sCD14) (R&D Systems, Minneapolis, MN, United States), and internal fatty acid-binding protein (iFABP) (MyBioSource, San Diego, CA, United States) in plasma samples were measured using an enzyme-immunoassay technique (ELISA) following the manufacturer’s protocol. Plasma levels of free bacterial endotoxin were measured using a chromogenic Limulus amebocyte lysate assay (Hycult Biotech, Uden, Netherlands) following the manufacturer’s protocol. Plasma levels of circulating immune factors were measured using a Magnetic 33-plex assay (R&D Systems, Minneapolis, MN, United States) and a MAGPIX instrument (Luminex, Austin, TX, United States). Bubble plots were generated using ggplot2 R package with concentration (pg/ml).

ELISAs for zonula occludens (ZO-1), Dickkopf-related protein-1 (DKK-1), and intestinal fatty acid binding protein (iFABP) were purchased from MyBioSource (San Diego, CA) and were used according to the manufacturer’s instructions. In brief, sera from all animals were stored in a freezer at –80°C and thawed immediately before use. The serum was added to the empty wells of ELISA plates containing standard reagents, and blank wells for controls were incubated with detection antibody and washed, detection was reagent added, and then plates were read at 420 nm on a Cytation 3 imaging reader (Biotek, Winooski, VT). Concentrations of samples were calculated according to the manufacturer’s (MyBioSource) formula.