Type 1500 multiskan spectrum microplate reader

To quantify the degree of infection-induced damage to the intestinal epithelium, the concentration of intestinal fatty acid-binding protein (I-FABP) was measured in fetal plasma samples by ELISA as previously described27 (link). Briefly, an Elisa plate was coated with 3 μg/well of anti-human I-FABP monoclonal antibody for 24 hours. A dilution series of a known concentration of human recombinant I-FABP protein was used for a standard curve. Plasma samples were diluted two times and were incubated with biotin-conjugated polyclonal anti-human I-FABP antibody, followed by incubation with streptavidin peroxidase. After incubation with the substrate, the reaction in the samples was terminated by adding 1M of H₂SO₄ and the optical density was measured at 450 nm in a Thermo Electron Type 1500 Multiskan Spectrum Microplate Reader.

Levels of the pro-inflammatory cytokines interleukin (IL)-6 and IL-8 were measured in fetal plasma as markers for systemic inflammation using ovine-specific sandwich enzyme-linked immunosorbent assays (ELISA) as previously described [8 ].
In short, a 96-wells plate was coated with a monoclonal mouse-anti IL-6 (Millipore Cat# MAB1004, working concentration 1:200) or IL-8 (Millipore Cat# MAB1044, working concentration 1:200) and incubated overnight at 4 °C. The standard curve and serum samples were diluted in PBS + 0.1% BSA in 1:1 or 1:80, respectively, for IL-6 and IL-8. Incubation with the detection antibody rabbit-anti-ovine IL-6 (Millipore Cat# AB1839, working concentration 1:500) or IL-8 (AB1040, Millipore, working concentration 1:500) was performed for 1 h, followed by incubation with a HRP-labeled antibody (Jackson ImmunoResearch Labs Cat# 111-035-045, working concentration 1:500). Next, incubation with 3,3′5,5′-tetramethylbenzidine (TMB) substrate solution was done for 10 (IL-6) or 2,5 (IL-8) minutes. The reaction was stopped by addition of H₂SO₄, and the optical density (OD) was measured at 450 nm in a Thermo Electron Type 1500 Multiskan Spectrum Microplate Reader. Concentrations were expressed relative to a standard curve of recombinant ovine IL-6 or IL-8 (ImmunoChemistry Technologies, Bloomington, MN, USA).