Human leptin immunoassay

Selected cytokines and adipokines were measured using the specific Quantikine^® ELISA kits (Human Leptin Immunoassay, Human Adiponectin Immunoassay, Human TNFα Immunoassay, Human IL-10 Immunoassay, and Human INFγ Immunoassay) purchased from R&D Systems (Minneapolis, MN, USA). All ELISA assays were performed on serum samples collected at T0 and T1, in the same batch, following the manufacturer’s instructions.

Blood was transferred into tubes containing 0.3 mol·L⁻¹ EDTA (10 μL·ml⁻¹ blood) and immediately centrifuged at 4°C for 10 min at 23,000 g. The plasma was stored at –80°C until analysis. Analysis of plasma insulin and glucose has been described previously (Ara et al., 2011 (link)). The Quicki index was calculated as previously described (Katz et al., 2000 (link)). Blood HbA1c was analyzed on a DCA Vantage Analyzer (Siemens Healthcare, NY, USA). Plasma IL-6 and plasma TNF-α were measured with high-sensitivity ELISA kits from R&D Systems (Minneapolis, MN, USA). The plasma IL-18 was analyzed with HS ELISA (R&D Systems) and CRP using a conventional high-sensitivity assay on a Cobas Hitachi (Roche, Indianapolis, USA). The plasma adiponectin concentration was determined with a human radioassay kit (EMD Millipore. Billerica, MA, USA). The plasma leptin concentration was measured by a high-sensitivity human ELISA kit (Human leptin Immunoassay, R&D systems).