Competitive elisa kit

Serum samples were obtained after centrifugation (1600× g at 4 °C for 15 min) and then stored at −80 °C. Serum concentration of irisin was measured by a competitive ELISA kit (Phoenix Pharmaceuticals, Burlingame, CA, USA; Cat. No. EK-067-29), following the manufacturer’s instructions. This kit has sensitivity 1.29 ng/mL and linear range 1.29–27.5 ng/mL, intra-assay coefficient of variation (CV) < 10%, and inter-assay CV < 15%. In this assay, the immunoplate is pre-coated with a secondary antibody, whose non-specific binding sites are blocked. The secondary antibody can bind to the Fc fragment of the primary antibody. This primary antibody’s Fab fragment then binds competitively both the biotinylated peptide and the targeted peptide in the standard curve samples and the unknown samples. The biotinylated peptide interacts with streptavidin-horseradish peroxidase (SA-HRP) which catalyzes the substrate solution. The intensity of the resulting yellow color is inversely proportional to the amount of irisin, measured by spectrophotometer (Eon; BioTek Instruments, Inc., Winooski, VT, USA).

We assessed levels of VIP using a commercially available competitive ELISA kit (Phoenix Pharmaceuticals), as previously described^{17 (link)}. Serum samples were freeze-dried and dissolved in ELISA buffer (2:1). Levels of VIP were determined applying the corresponding dilution factor. Samples from each patient were assayed twice. The minimum detectable concentration was 0.12 ng/ml, with an intra-assay and inter-assay variation of ≤ 5 and 15%, respectively.

Mouse myoblast C2C12 cells were used for in vitro experiments in this study. Myoblast were plated at 10³ cells/cm² and cultured in Minimum Essential Medium Eagle–Alpha Modification (α-MEM) (Gibco; Thermo-Fisher, Waltham, MA, USA) with 10% fetal bovine serum (Gibco; Thermo-Fisher, Waltham, MA, USA) until they reached confluence in a humidified atmosphere (37 °C, 5% CO₂) (Hera cell 150; ThermoFisher, Waltham, MA, USA). For knocking down Sirt1, C2C12 myoblasts were transfected with Sirt1 siRNA (80 nM) or Scramble (scr) using Lipofectamine RNAiMAX Reagent (Invitrogen, ThermoFisher, Waltham, MA, USA) and then treated with 10⁻⁸ M of vitamin D for 48 h.
The concentration of irisin in the cell culture media was measured using a competitive ELISA kit (Phoenix Pharmaceuticals, Burlingame, CA, USA, Cat. No. EK-067-29), which allowed for the detection of a very low concentration of protein (ng/mL). This kit had a sensitivity of 1.29 ng/mL, linear range 1.29–27.5 ng/mL, intra-assay coefficient of variation (CV) of <10%, and inter-assay CV of < 15%.