Fasting blood samples were drawn from antecubital veins of the participants into plain glass tubes while they were in sitting position. After collection, samples were spun for 8–10 min at 1500 xg to obtain serum for BDNF. Serum samples were then divided into several aliquots and immediately stored at -80 °C for future use.
Serum BDNF level was determined using enzyme linked immunosorbent assay (ELISA) specific for BDNF and as described in the kit manual (Human BDNF Duoset ELISA Kit R&D system, USA). In brief, the samples were added to an anti-human BDNF antibody coated ELISA wells and allowed to incubate for 2 h at room temperature. Subsequently, biotinylated anti-human BDNF antibody was introduced and allowed to bind to the captured BDNF for 1 h at the same temperature. Horse-radish-peroxidase (HRP)-streptavidin conjugate was afterward added to the reaction mixture then incubated at room temperature for 45 min. Finally, tetramethyl benzidine (TMB) was added to each well followed by additional incubation for 30 min at room temperature in the dark. The color developed after the incubation was read at 450 nm immediately and levels of serum BDNF were deducted from the standard provided by the kit [14 (link), 24 (link)].
Serum BDNF level was determined using enzyme linked immunosorbent assay (ELISA) specific for BDNF and as described in the kit manual (Human BDNF Duoset ELISA Kit R&D system, USA). In brief, the samples were added to an anti-human BDNF antibody coated ELISA wells and allowed to incubate for 2 h at room temperature. Subsequently, biotinylated anti-human BDNF antibody was introduced and allowed to bind to the captured BDNF for 1 h at the same temperature. Horse-radish-peroxidase (HRP)-streptavidin conjugate was afterward added to the reaction mixture then incubated at room temperature for 45 min. Finally, tetramethyl benzidine (TMB) was added to each well followed by additional incubation for 30 min at room temperature in the dark. The color developed after the incubation was read at 450 nm immediately and levels of serum BDNF were deducted from the standard provided by the kit [14 (link), 24 (link)].