Quantifying BPDE-DNA Adducts in Villous Tissue

Genomic DNA was isolated from human villous tissues using Tissue/Cell Genomic DNA extraction and Purification Kit (K1442-100; BioVision). The levels of BPDE-DNA adducts were assessed using BPDE-DNA adduct ELISA kit (STA-357; Cell Biolabs). Briefly, DNA samples ( $2\mathrm{\mu }\mathrm{g}/\mathrm{mL}$ ) were sonicated into fragments with 200–1,000 bp as determined by 1% agarose gel and then incubated with anti-BPDE antibody (dilution 1:1,000; 235601, Cell Biolabs) in 96-well plates for 2 h at room temperature. After washing with wash buffer (310806; Cell Biolabs), the secondary antibody (10902; Cell Biolabs) was added to each well for 1 h, 3,3′,5,5′-tetramethylbenzidine buffer was added, and then the mixture was incubated for 20 min at room temperature. After termination of reaction, the relative levels of BPDE-DNA adducts were determined by measuring the absorbance at $450\text{\hspace{0.17em}nm}$ using a microplate reader (VL0L00D0; Thermo Fisher Scientific), with the Reduced DNA Standard (235602; Cell Biolabs) as absorbance blank. The amount of BPDE-DNA adducts was quantified using a BPDE-DNA standard curve. The results were expressed as nanograms of BPDE-DNA adducts per microgram of DNA.