Quantikine hs elisa

Plasma samples and supernatants from PBMC stimulations were analyzed using commercially available ELISA kits according to the manufacturers' instructions. In detail, plasma samples were analysed for IL-6 (Quantikine HS ELISA; R&D Systems Europe, Wiesbaden, Germany), and cortisol (IBL International, Hamburg, Germany). As TSST-induced changes in plasma cortisol in the original study (8 (link)) were most pronounced between time point 1 (baseline; –5 min) and 3 (+15 min), we used the respective AUC to compare urban participants who grew up with absolutely no animal contact with urban participants who grew up with occasional animal contact (Figure 3) in the present study. Accordingly, we calculated AUC between time point 5 (90 min) and 6 (120 min) for plasma IL-6 levels in the current study to compare urban participants who grew up with absolutely no animal contact with urban participants who grew up with occasional animal contact (Figure 3), as TSST-induced differences between urban and rural participants in plasma IL-6 levels were only detectable at these late stages.

Venous blood was collected from an antecubital vein using a 21-gauge needle into an 8 ml gel separator tube. All samples were subsequently centrifuged at 10 °C for 10 min at 3000 rpm, and stored in 1.5 mL eppendorfs at −80 °C until further analysis at a commercial pathology laboratory (PathWest Laboratory, Fiona Stanley Hospital). The IL-6 was analysed via immunoassay technique (Quantikine HS ELISA, R&D Systems, Inc. Minneapolis, USA). The coefficient of variation (CV) for inter-assay precision at 0.49 and 2.78 pg/mL was 9.6 and 7.2% respectively. The hsCRP was measured using an Architect analyser (ci8200), and determined using a CRP Vario Reagent (SENTINEL CH. SpA, Via Robert Koch, 2, Milan 20152, Italy). The CV for CRP determination at 0.88, 2.21 and 6.22 mg/L was 2.3, 1.2 and 1.0%, respectively. The UA was measured using an Architect analyser (ci8200), and determined using a UA Reagent (Abbott Diagnostics, Abbott Laboratories, Abbott Park, IL 60064, USA). The CV for UA determination at 0.25 and 0.56 mg/L was 1.92 and 1.5%, respectively. F₂-IsoP was analysed using an Agilent 6890 gas chromatograph coupled to an Agilent 5973 mass selective detector. The mean total (free + esterified) plasma F₂-IsoP concentration was 952 ± 38 pmol/L, with a within and between assay reproducibility of 8.0 and 5.6%, respectively [16 (link)].

ELISA was performed on 50 μl of SK-OV-3 cell media or patient serum samples using commercially available reagents to measure human TARS (Cusabio Biotech) or TNF-α (R&D Systems Quantikine® HS ELISA). Values were determined by comparing to a standard curve (0–800 pg/ml for TARS, 0–32 pg/ml for TNF-α).

For the screening of PPP blood levels, the following immune mediators were individually quantified by enzyme-linked immunosorbent assays (ELISAs): lipocalin-2, β-defensin 2, IL-22, IL-20, IL-19, IL-1β, TNF-α, serum amyloid A (SAA), CCL2, CXCL6, resistin, chemerin, fetuin-A, and angiogenin. For SAA and β-defensin 2, ELISAs from Invitrogen and Immundiagnostik AG were used, respectively; all other assays were Quantikine™ ELISA systems obtained from R&D Systems (high sensitivity Quantikine™ variants for IL-1β and TNF-α). Quantification of β-defensin levels in serum obtained from PPP and PsV patients (as confirmation of in vitro results) was performed using Quantikine™ HS ELISA from R&D Systems. Blood samples from PPP patients participating in the APLANTUS study were subjected to Quantikine™ ELISAs from R&D Systems to individually quantify the following immune mediators: IL-19, IL-22, G-CSF, LCN2, MMP8, sP-selectin, SCF, sSCF-R.