Varioskan flash spectral scanning multimode reader

A commercial Human Interleukin 2 Receptor α (IL-2Rα/CD25) kit (Elabscience) was used according to the manufacturer's instructions. In total, ~100 µl of standard and samples were added to the precoated ELISA plate. The plate was precoated with an antibody specific to CD25. In total, ~100 µl of biotinylated detection antibodies specific to human CD25 and 100 µl of avidin-horseradish peroxidase (HRP) conjugates which were part of the kit were successively added to each microplate well and incubated for 90 min respectively at 37°C. Next, the unbound components were washed away, and the substrate was added. The enzyme-substrate reaction was stopped by the addition of 50 µl of stop solution. The optical densities were measured spectrophotometrically at a wavelength of 450 nm using the Varioskan Flash spectral scanning multimode reader (Thermo Fisher Scientific Inc.). The CD25 concentration was calculated using the standard curve.

The height and weight of patients were measured by the same trained group. The body mass index (BMI) was calculated as the weight in kilograms divided by the height in meters squared. In our previous study, serum irisin levels were accurately measured using a highly quantitative enzyme-linked immunosorbent assay (ELISA) kit from Phoenix Pharmaceuticals Science, Inc. (Burlingame, CA, USA). An automated ELISA reader was used to quantify the results (Varioskan Flash Spectral Scanning Multimode Reader; Thermo Fisher Scientific, Waltham, MA, USA). Irisin levels in the two measurements were consistent.^{23 (link)} Therefore, in the present study, serum irisin levels were measured using an ELISA kit. CHOL, HDL-C, LDL-C, TG, CK, and FPG levels were measured using the Dade-Behring Dimension RXL Autoanalyzer (Siemens Healthcare Diagnostics, Marburg, Germany). The reference intervals for CHOL, HDL-C, LDL-C, TG, CK, and FPG were 3.62 to 5.70 mmol/L, 1.03 to 1.55 mmol/L, 1.81 to 3.36 mmol/L, 0.56 to 2.26 mmol/L, 38 to 174 U/L, and 3.3 to 6.1 mmol/L, respectively. FT3, FT4, and TSH levels were measured by an electrochemiluminescence immunoassay technique using the Abbott Architect i2000 (Abbott Diagnostics, Abbott Park, IL, USA). The reference intervals for FT3, FT4, and TSH were 1.71 to 3.71 pg/mL, 0.70 to 1.48 ng/dL, and 0.35 to 4.94 µIU/mL, respectively.

The TNF-α and histamine concentrations in serum were measured via ELISA in accordance with the instructions indicated in the TNF-α ELISA kit (RTA00, R&D Systems, USA) and histamine ELISA kit (LS-F27982-1, LifeSpan Biosciences, Seattle, USA), respectively. The TNF-α and histamine concentrations were measured at 450 nm using a Varioskan® Flash Spectral Scanning Multimode Reader (Thermo Fisher Science, Waltham, MA, USA).