Quantikine human immunoassay

Quantikine Human Immunoassays (R&D, Minneapolis, MN, USA) were used to quantify serum concentrations of I-FABP, IL-6 and IL-10. Immunoturbidometry was used to analyze serum immunoglobulin concentrations.

Blood samples were collected from seated patients after a 10-h overnight fast and examined to determine HbA1c (%), fasting plasma glucose (mg/dl), TC (mg/dl), HDL-C (mg/dl), LDL-C (mg/dl), TG (mg/dl), uric acid (mg/dl), activin A (pg/ml), and follistatin (pg/ml) concentrations. Serum concentrations of glucose, TC, TG, LDL-C, HDL-C, and insulin were determined using an automatic analyzer (ADVIA 1800; Siemens, Malvern, PA, USA). Serum insulin-like growth factor-1 concentrations were measured by enzyme-linked immunosorbent assay (ELISA) (Ray Biotech, Norcross, GA, USA). The whole-blood HbA1c concentration was measured using an enzymatic method with a high-performance liquid chromatography analyzer (G8; Tosoh Bioscience, Inc., San Francisco, CA, USA). A single voided morning urine sample was used to measure the urinary albumin-to-creatinine ratio^{40 (link)}. Activin A and follistatin concentrations were measured by ELISA (Quantikine human immunoassays; R&D Systems, Minneapolis, MN, USA). All serum samples were tested in duplicate, and coefficients of variation of duplicate samples were less than 20%.

The Quantikine^® Human Immunoassays (all from R&D Systems, Inc., Minnesota, CO, USA) were used to quantify soluble AXL (cat. No. DAXL00), ICAM1 (cat. No. DCD540), IL6Rα (cat. No. DR600) and IL8 (cat. No. D8000C) in cell culture supernatants from treated or not cell lines, according to manufacturer’s instructions. The optical density per well was measured at 450 nm and 570 nm to correct optical imperfections using a Multiskan EX Microplate Photometer (Thermo Scientific, Courtaboeuf Cedex, France).