Immuno-slot-blot: A highly sensitive immunoassay for the quantitation of carcinogen-modified nucleosides in DNA

Publisher Website
Google Scholar
Abstract
We have established a highly sensitive immuno-slot-blot (ISB) procedure that can be routinely applied for detection and quantitation of any heat- or alkali-stable structural DNA modification (caused by carcinogens or mutagens, for example) for which a specific (monoclonal) antibody (MAB) is available. The essential step in this assay is the immobilization on nitrocellulose filters of the structurally modified DNA in its single-stranded form. The immobilized DNA is first reacted with an MAB specifically directed against a particular modified DNA component (e.g., an alkyldeoxynucleoside), and thereafter with a second antibody directed against the first one. The second antibody can be either labeled with 125I or linked to an enzyme complex capable of eliciting a color reaction with a suitable substrate. The sensitivity of the ISB is demonstrated for two different alkyldeoxynucleosides, O6-ethyldeoxyguanosine (O6-EtdGuo) and O4-ethyldeoxythymidine (O4-EtdThd), both of which are produced in cellular DNA exposed to the alkylating N-nitroso carcinogen N-ethyl-N-nitrosourea and both of which represent DNA lesions miscoding during DNA replication and transcription. Using anti-(O6-EtdGuo) and anti-(O4-EtdThd) MABs, respectively, O6-EtdGuo and O4-EtdThd are detected at levels as low as ≧0.3×10-15mol O6-EtdGuo/3 μg DNA (O6-EtdGuo/deoxyguanosine molar ratio in DNA, ≧2×10-7) and ≧0.1×10-15 mol of O4-EtdThd/3 μg DNA (O4-EtdThd/deoxythymidine molar ratio in DNA, ≧4×10-8).