Objective        To establish an easy method to detect the content of O6-alkyltransferase (O6 Methylguanine-DNA Methyltransferase

MGMT) constituted DPC

the MGMT-DNA crosslinkings (M-DPC).   Methods      Contemporary techniques was adopted and applied to some critical steps

such as M-DPC extraction and purification

M-DPC quantification

and optimizing the binding ability of M-DPC antigen to ELISA plate. Human bronchial epithelial cell line HBE was exposed to different concentrations of nitrogen mustard

and harvested at indicated time points. M-DPC samples were prepared by employing nucleus extraction

lysis and Tris-saturated phenol recovery. The dsDNA in M-DPC samples was determined by SYBR Green dyeⅠ. To enhance the binding ability of the antigen in M-DPC samples to the bottom of ELISA plate

universal nucleic acid enzyme (Benzonase) was employed to degradate the nucleic acid in M-DPC samples. Finally

the M-DPC coated plate was subjected to the conventional processes of standard ELISA.   Results      Cellular M-DPC was successfully extracted and purified independent on high speed centrifugation and radioactive isotope labeling. The linear range of SYBR Green Ⅰ method to detect genomic dsDNA was between 2 ng to 1.5 μg

which was suitable for detecting the DNA content in M-DPC samples

and based on which equal amount of samples was adjusted and applied. The M-DPC content in sample could be detected by standard ELISA method

in a linear relationship within a certain range of M-DPC concentration. M-DPC content was increased significantly in 3 h after nitrogen mustard treatment in a dose-dependent manner

and was maintained at a high level till 24 h.   Conclusion      Our method to detect M-DPC by ELISA is easy

rapid

sensitive

and high throughput

and may also be applied to detect DPC containing other proteins. Nitrogen mustard-induced M-DPC is in a time- and dose-dependent manner.