Detection of inflammatory and epigenetic markers
Serum levels of IL-6 (Gen-Probe, Diaclone Diagnostic, France), IL-17 (Gen-Asia Biotech, China), IL-10 (Bioassay Tech Laboratory, China), and HDAC1 (Qayee Bio-Technology) were estimated by ELISA using commercially available kits. Briefly, the workflow of the HDAC1 ELISA kit utilizes the sandwich ELISA methodology where the plate was pre-coated with human HDAC1 antibody. The serum samples were then added onto the pre-coated wells. And, then biotinylated human HDAC1 antibody was added, which binded to HDAC1 in the sample. After the addition of Streptavidin-HRP developer followed by substrate and stop solution, the well absorbance for HDAC1 was measured at 450 nm. The 5-methylcytosine DNA ELISA kit (Enzo Life Sciences, Inc., USA) and 5-hydroxymethyl cytosine DNA ELISA kit (Enzo Life Sciences, Inc., USA) was used to quantify the percent 5-mC DNA and 5-hmC DNA respectively. Briefly, the workflow for the 5-mC DNA ELISA kits utilize the indirect ELISA methodology where 100 ng denatured, single-stranded DNA (ssDNA) samples per well were coated on the plate wells and a 5-mC mAb and conjugate HRP-Ab were then added to the wells. The detection of 5-mC was done after the addition of the HRP developer by measuring well absorbance at 405–450 nm. Briefly, the workflow of the 5-hmC DNA ELISA kit utilizes the sandwich ELISA methodology where a 5-hmC pAb was coated to the bottom of plate well surfaces. The ssDNA (100 ng/well) sample was then added onto the well surface which binded to 5-hmC pAb’s and was then recognized by a conjugate DNA HRP-Ab. After the addition of HRP developer, the well absorbance for 5-hmC DNA was measured at 405–450 nm. Serum TGF-β levels were estimated by a magnetic bead-based multiplex assay using Bio-Plex Pro TGF-β Assays (Bio-Rad Laboratories Inc., USA) according to the manufacturer's guidelines. Quality-control assays for biomarkers and validation were performed.