2.6. Endotoxemia
LBP
SECONDARY
Serum endotoxin was measured using a fluorometric PyroGene™ recombinant factor C assay (Lonza (Basel, Switzerland), 50-658U). The assay is based on endotoxin-mediated activation of recombinant factor C, which then cleaves a fluorogenic compound that can be monitored at 380/440 nm (excitation/emission). In brief, serum was diluted 1:100 in endotoxin-free water, incubated (10 min, 70 °C), and mixed with kit reagent prior to monitoring fluorescence at 0 min and 60 min following incubation (37 °C). Quantification was based on the net fluorescence of samples against that of endotoxin standards prepared in parallel. Intra- and inter-assay CVs were 2.6% and 10.6%, respectively. Plasma LPS-binding protein (LBP; DY870) and soluble CD14 (sCD14; DC140) were measured using separate ELISA kits (R&D Systems, Minneapolis, MN, USA) as secondary measures of endotoxin exposure [