2.5. BPDE-DNA Adduct Formation Analysis

DNA extraction was performed using a QIAamp DNA Mini Kit (Qiagen, Valencia, CA, USA), according to the manufacturer’s instructions. DNA was isolated and the level of BPDE-DNA adduct formation was assessed using BPDE-DNA adduct ELISA kit (Cell Biolabs, San Diego, CA, USA) and following manufacturer’s instructions. Briefly, DNA samples of 2 µg/mL concentration are prepared. An amount of 100 µL of each sample is treated in 96 well plates for 2 h at 37 °C and rinsed two times. After washing, 100 µL of anti-BPDE antibody is treated in each well and incubated for 2 h at room temperature. Each well is rinsed 5 times using washing buffer, and then secondary antibody is added in each well for 1 h at room temperature. Each well is rinsed 3 times using washing buffer. All wells are reacted with 3,3′,5,5′-Tetramethylbenzidine (TMB) buffer at room temperature for 20 min. Finally, the reaction is stopped by adding stop solution. The BPDE-DNA adducts formation level was evaluated by measuring the relative absorbance using a microplate reader at 450 nm.