High sensitivity elisa

One blood sample was collected per participant into an anticoagulated (EDTA) tube at 8 a.m. on one morning of the study. Samples were centrifuged at 4°C, and aliquots of plasma were prepared for storage within 30 min of collection, and then immediately frozen at −70°C until assays were conducted. At the end of the studies, plasma samples were thawed and assayed for inflammatory markers. Each sample was assayed in duplicate using enzyme-linked immunosorbent assays (ELISAs), according to manufacturer's instructions except as noted. CRP levels were determined by a high sensitivity ELISA (Immundiagnostik, ALPCO Immunoassays, Salem, NH) at a routine sample dilution of either 1:200 or 1:500 (up to 1:2000 as needed), and with an extended standard curve, for an assay range of 0.2–300 mg/L (taking the sample dilution into account). Plasma levels of TNFα and IL-6 were determined by Quantikine high sensitivity ELISAs (R&D Systems, Minneapolis, MN); IL-6 samples were routinely diluted 3-fold, and when necessary, further diluted up to 20-fold in order to measure samples with IL-6 concentrations up to 200 pg/mL (taking sample dilution into account). Measurement of sICAM-1 was performed using a regular sensitivity sICAM-1 ELISA (R&D Systems, Minneapolis, MN). For all four inflammatory markers, intra-assay variability was less than 6% and inter-assay variability was less than 8%.