Cortisol elisa kit

Blood was collected (3 mL; sodium heparin vacutainer tubes; Becton, Dickinson and Company, Franklin Lakes, NJ, USA) 1 day prior to transport (pre-transport) at approximately 1500 h and immediately after transport (post-transport) at approximately 1600 h. Blood was not collected for the remaining 7 day of the study so as not to disturb the natural behavior of the piglets and influence behavioral data collection. Samples were centrifuged (4 °C, 1900× g for 15 min), and then plasma was collected, aliquoted, and stored at −80 °C. Commercially available ELISA kits were used according to manufacturer’s instructions to measure plasma cortisol (Cortisol ELISA Kit; Enzo Life Sciences, Inc., Farmingdale, NY, USA) and insulin (Mercodia Porcine Insulin ELISA; Mercodia AB, Uppsala, Sweden) concentrations. A commercially available colorimetric assay was used to measure plasma glucose (Autokit Glucose; Wako Pure Chemical Industries, Ltd.; Chuo-Ku Osaka, Japan) and non-esterified fatty acid (NEFA) concentrations (HR Series NEFA-HR (2); Wako Pure Chemical Industries, Chuo-Ku Osaka, Japan) according to manufacturer’s instructions. The intra-assay and inter-assay coefficient of variation for cortisol, NEFA, glucose, and insulin were 4.2, 2.0, 2.3, 4.8 and 2.4, 2.9, 4.4, 6.5%, respectively.

The cortisol was analyzed with plasma using a Cortisol ELISA kit (ADI-900-071, Enzo Life Science, New York, NY, USA) following the manufacturer’s protocols. The sensitivity of the kit was 56.72 pg/mL. Samples were diluted with a steroid displacement reagent at a ratio of 1:100 and analyzed in duplicate. A microplate reader (Tecan, Switzerland) calculated the concentration of cortisol, 5-HT, and β-endorphin in the samples based on an absorbance of a wavelength of 405 nm. The average intra- and inter-assay coefficients of variation were less than 10.5 and 13.4%, respectively.

Cortisol metabolite levels were measured using a Cortisol ELISA kit (Enzo Life Sciences^®, ADI-900-071, New York, NY, USA) that has broad cross-reactivity and has previously been validated for faecal samples assessment in other mammal species [37 (link),38 (link)], including primates [39 (link)]. According to the manufacturer, the cross-reactivity was cortisol (100%); prednisolone (122.35%); corticosterone (27.68%); 11-deoxycortisol (4.0%); Progesterone (3.64%); prednisone (0.85%); testosterone (0.12%) and <0.10% with androstenedione, cortisone, and estradiol. The sensitivity of the assay was 56.72 pg/mL (range 156–10,000 pg/mL). Samples were diluted 1:10 with the assay buffer. All faecal samples and standards were assayed in duplicate. Assay data were analysed utilising a 4-parameter logistic (4PL) fitting programme (MyAssays^®, Brighton, UK). Intra-assay coefficients of variation at low, medium, and high concentrations of cortisol were 10.5%, 6.6%, and 7.3%, respectively; inter-assay coefficients at low, medium, and high concentrations of cortisol were: 13.4%, 7.8%, and 8.6%, respectively.

The subject's cortisol and sympathetic hormone levels were used as indexes of stress-related hormonal responses after experiencing nine sessions of social interactions. The batch #2 subjects (n = 8 per group) were used in this experiment. Blood samples were collected from the trunk of the animals and centrifuged at 3000 rpm at 4°C for 20 min. Sera were divided from blood and then stored at –80°C until use. The cortisol assay was performed using a cortisol ELISA kit (Enzo Life Sciences, Farmingdale, NY, U.S.A.), and the experimental procedure was modified from Wommack and Delville (2003 (link)). The ELISA plate was measured at 405 nm using a Multiskan FC microplate photometer (Thermo Fisher Scientific, Waltham, MA, U.S.A.), and the intra-assay variability was 15.6%. The cortisol level is presented as a concentration (ng/ml). The serum adrenaline and noradrenaline levels were measured using the 2-CAT (A-N) Research ELISA Kit (Labor Diagnostika Nord, Nordhorn, Germany). The adrenaline and noradrenaline ELISA plates were measured at 450 nm and a reference wavelength of 620 nm using a Multiskan FC. The intra-assay variability was 8.5% for adrenaline and 13.6% for noradrenaline. The levels of sympathetic hormones are presented as concentrations (ng/ml).