Elisa bdnf emax immunoassay system

Mature BDNF protein level was measured using the BDNF Emax ImmunoAssay (ELISA) system (Promega), with recombinant mature BDNF as a standard. Standard and samples were performed in duplicate, with each group containing 10–14 samples. Protein was extracted and quantified following the manufacturer’s protocol. Tissue samples were homogenized in lysis buffer (150 mm NaCl, 1% Triton X-100, 25 mm HEPES, 2 mm NaF) containing phosphatase and protease inhibitors, and then incubated by rotation at 4°C for 1 h. Homogenized tissue was centrifuged at maximum speed and the supernatant containing total protein was collected and quantified using the BCA protein assay kit (Thermo Fisher Scientific). Each sample was diluted 1:1 with block and sample buffer (BSB), and placed in designated wells of a 96-well plate previously coated with BDNF antibody in carbonate buffer (25 mm Na₂CO₃ and 25 mm Na₂HCO₃, pH 9.7, incubated at 4°C), followed by blocking with BSB. A second coating of primary anti-human BDNF antibody was added, followed by horseradish peroxidase-conjugated secondary antibody. The colorimetric reaction was initiated by tetramethylbenzidine. After 10 min, the reaction was stopped by addition of 1N HCl, and absorbance was read at 450 nm on a plate reader (iMark Absorbance Microplate Reader, Bio-Rad Laboratories).