High sensitivity salivary cortisol eia kit

Serum samples from all animals were analysed for haptoglobin, C-reactive protein (CRP) and Pig-MAP, and glutathione peroxidase (GPx). Haptoglobin was determined spectrophotometrically (Phase Haptoglobin, Tridelta Ltd, County Kildare, Ireland). CRP was determined with an immunoturbidimetric method (OSR #6147, Winston-Salem, NC, USA) validated for porcine samples [35 (link)]. Pig-MAP was measured by ELISA (PigChamp ProEuropa, Segovia, Spain). GPx was determined with the Cumene Hydroperoxide method (Ransel, Randox Laboratories Ltd, Crumlin, UK). Superoxide dismutase (SOD) was determined in PBMC lysates with the Xanthine oxidase method (Ransod, Randox Laboratories Ltd, Crumlin, UK). All techniques were adapted to the Olympus AU400 analyser. Cortisol concentrations were determined using commercial ELISA kits, namely DRG Cortisol ELISA (DRG Diagnostics, Marburg, Germany) for serum samples, DRG Salivary Cortisol ELISA (DRG Diagnostics, Marburg, Germany) for salivary samples and High Sensitivity Salivary Cortisol EIA Kit (Salimetrics, State Collage, PA, USA) for hair samples. Protocol for hair extraction and assay validation are described in [36 ].

Salivary samples were collected three times under a free-living setting using a Salimetrics oral swab (Salimetrics Europe, Suffolk, UK), i.e., a small pad, absorbent and nontoxic, passed within the oral cavity for 2–3 min before being placed inside a labeled tube. The samples were stored at 8 a.m., 5 p.m., and 11 p.m.; refrigerated within 30 min; and frozen at −20 °C within 2 h of collection. On the day of the assay, samples were thawed and centrifuged for 15 min at 3000 rpm to extract saliva and remove the mucin, and the swab was then discarded. Each sample was processed in duplicate. Salivary samplings from 8 a.m. were collected in fasting condition, except for water consumption; in contrast, those from 5 p.m. and 11 p.m. were collected 2 h after any food or beverage consumption, except for water. The sum of the three samplings results was considered representative variable data of daily salivary cortisol excretion [44 (link)]. The assays were performed using the High Sensitivity Salivary Cortisol EIA kit and Salivary DHEA-S EIA kit (Salimetrics Eu-rope, Suffolk, UK), with an intra-assay coefficient of variation of 4.6% for cortisol and 7.25% for DHEA-S and an interassay precision of 6% for cortisol and 7.5% for DHEA-S.