Elisa

Postoperatively, the body weights were monitored weekly. Serum lipid profiles, including total cholesterol, triglyceride and free fatty acid (FFA) were measured using enzyme-linked immunosorbent assay (ELISA, R&D Systems, Minneapolis, Minnesota, USA).
Plasma insulin was quantified using ELISA, in accordance with the manufacturer’s instructions (R&D Systems). Prior to operating, and at 2 and 8 weeks postoperatively, homeostasis model assessment of insulin resistance (HOMA-IR) was calculated to evaluate insulin resistance according to the following formula: HOMA-IR = fasting insulin (mIU/L) × fasting glucose (mmol/L)/22.5.
Plasma levels of IL-6, IL-1β, monocyte chemotactic protein-1, TNF-α and FGF21 were assayed with ELISA, in accordance with the manufacturer’s instructions (R&D Systems).

We measured IL-1β, TNF-α, IL-6, and IL-10 in these 66 samples using ELISAs (Bio-Techne, formerly R&D systems, Minneapolis, MN) and studied the operation characteristics of ELISAs from our own experiments. Although we did not measure IL-1β, TNF-α, IL-6, and IL-10 in these 66 samples using the Bio-Plex^® and Multi-Array assays, we included the operational characteristics of the BioPlex^® and Multi-Array assays, which were derived from our previous experiences in using these systems [23 (link)–25 (link)], in the comparison to those of the Simple Plex and ELISAs.

For plasma cytokine concentrations, EDTA anticoagulated blood was centrifuged (2,000 g, 4°C, 10 min) and stored at −80°C until analysis. Concentrations of cytokines in plasma, nasal wash, and supernatants of stimulated monocyte cultures were determined by simultaneous Luminex assay (R&D Systems; Abingdon Science Park, UK) and enzyme-linked immunosorbent assays (ELISAs) (R&D Systems, Minneapolis, MN, USA). TNF-α, IL-6, and IL-10 were measured in plasma samples collected on day 0 using a simultaneous Luminex assay (R&D Systems; Abingdon Science Park, UK). In samples obtained from day 7 onward, G-CSF, IL-6, IL-8, IL-10, and IFN-γ in nasal wash were measured using a Luminex assay from R&D Systems (Minneapolis, MN, USA), and IFN-α and IFN-β were measured by a Luminex assay from eBioscience (Vienna, Austria). IP-10 concentrations in nasal wash and plasma were measured using an ELISA (R&D Systems, Minneapolis, MN, USA). Lower detection limits in plasma were 1.2 pg/ml for TNF-α, IL-6, and IL-10, and 156 pg/ml for IP-10. In nasal wash, lower detection limits were 309 pg/ml for IP-10, 0.49 for IFN-α and IFN-β, and 1.4 pg/ml for the remaining analytes. Cytokines in supernatants of ex vivo stimulated monocytes were measured using ELISA (IL-1β and IL-13: R&D Systems, Minneapolis, MN, USA, IL-6 and IL-10: Sanquin, Amsterdam, The Netherlands) following the protocols of the manufacturers.

The levels of pro‐MMP‐1, pro‐MMP‐13, and total‐MMP‐3 were detected in cell culture supernatants of Gal‐1‐ or Gal‐3‐treated IVD cells (all ELISAs; R&D Systems, Minneapolis, MN). Supernatants of untreated IVD cells served as controls. Also, supernatants of untreated IVD cells were processed for galectin secretion (ELISAs from R&D Systems). Ranges of standard curve were 0.313–20 ng/ml for Gal‐1, 0.157–10 ng/ml for Gal‐3, 0.157–10 ng/ml for pro‐MMP‐1 and total‐MMP‐3, and 78–5,000 pg/ml for pro‐MMP‐13.

C-reactive protein was measured using an enzyme-linked immunosorbent assay (ELISA) (MP Biomedicals, Santa Ana, CA) with sensitivity and coefficient of variation (CV) at 2.81 ng/ml and <9.2% respectively. Total and high molecular weight adiponectin were analyzed using ELISAs (R&D systems, Inc. Minneapolis, MN) with sensitivity and CV at 0.79 ng/ml and <6.9% and 0.195 ng/ml and <8.6% respectively. Free fatty acids were measured using enzyme-based methods and quantified by colorimetric assays (Wako Diagnostics, Richmond, VA) with sensitivity of 0.0014 mEq/L and CV <2.7%. Sandwich ELISA kits (R&D Systems, Inc. Minneapolis, MN) were used to measure interleukin -6, interleukin-10, and tumor necrosis factor -alpha (TNF-α) with sensitivity and CV of 0.70 pg/ml and <6.4%, 3.9 pg/ml and <7.5% and 1.6 pg/ml and <5.2% respectively. Leptin, was also analyzed using ELISA (R&D Systems, Inc. Minneapolis, MN) with sensitivity and CV at 7.8 pg/ml and <5.4% respectively.