Immunoassay

Maternal serum levels of PlGF, sEng, and sVEGFR-1 were determined by immunoassays (R&D Systems Inc., Minneapolis, MN) utilizing the quantitative sandwich enzyme immunoassay technique. Laboratory testing was conducted at the research core laboratory of the Perinatology Research Branch, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Department of Health and Human Services. Standard serum specimens were used in each laboratory run and analyte value was determined by interpolation from standard curves. The assays utilized 120 μL, 25 μL, and 25 μL of serum for the PlGF, sEng, and VEGFR-1 assays, respectively. The inter- and intra-assay coefficients of variation obtained in the laboratory were 4.6% and 3.6% for PlGF, 5.3% and 4.3% for sEng, and 2.8% and 2.7% for sVEGFR-1, respectively. The sensitivity of the assays was 5.7 pg/mL for PlGF, 40.0 pg/mL for sEng, and 12.9 pg/mL for sVEGFR-1.

Hs-CRP was measured by an immunoturbidometric assay with the use of reagents and calibrators from Dade Behring Marbura GmbH (Marburg, Germany; interassay CV < 5.0%, CV: coefficients of variance). TNF-α was measured by immunoassays (R&D Systems, Inc., Minneapolis, MN; interassay CV = 6.0%). PAI-1 was measured by an ELISA method (Mitsubishi Chemicals; interassay CV = 8.1 %). For the purpose of statistical analysis, serum concentrations of hs-CRP and TNF-α below the limit of detection were assigned a value of 0.05mg/L and 0.50pg/mL (the lowest limit of detection), respectively. Systemic oxidative stress was evaluated by urinary creatinine-indexed 8-epi-prostagland in F-2α (8-epi-PG2α), a validated biomarker of oxidative stress [13 (link)]. Urinary 8-epi-PGF2α was measured in the first-voided morning urine sample with an enzyme-liked immunosorbent assay (8-Isoprostane EIA kit, Cayman, Ann Arbor, MI). Intra- and inter- assay CV were 7.5% and 9.2%, respectively. Urinary 8-epi-PG2α was indexed to creatinine as pictograms per millimole creatitine. Adiponectin was assayed by a sandwich enzyme-linked immunosorbent assay (Otsuka Pharmaceutical Co. Ltd., Tokushima City, Japan). Intra- and inter- assay CV were 3.3% and 7.5%, respectively. Leptin was assessed by an RIA kit (LINCO research, St. Charles, MO; interassay CV = 4.9%).