Detection of DNA Damage in Cells Exposed to Ionizing Radiation by Use of Anti-single-stranded DNA Monoclonal Antibody

Abstract

An immunochemical method has been developed for quantitative detection of DNA damage in mammalian cells. The method is based on the binding of a monoclonal antibody to single-stranded DNA. The clone producing this antibody (D1B) was obtained as a by-product from fusion of mouse myeloma cells with spleen cells isolated from a mouse immunized with chemically modified DNA. The technique is based upon the determination of the percentage single-strandedness resulting from the time-dependent partial unwinding of cellular DNA under alkaline conditions. Single- and double-strand DNA breaks, or lesions converted into such breaks in alkaline medium, form initiation points for the unwinding. The extent of unwinding from these points under defined conditions is a measure of the number of such sites. The method is rapid, does not require radioactive labelling of DNA or physical separation of single- from double-stranded molecules, is sufficiently sensitive to detect damage induced by 1 Gy of ionizing radiation and needs only small numbers of cells. The usefulness of the technique was demonstrated in a study of the induction of DNA damage and its repair in cultured Chinese hamster cells and in human white blood cells after exposure to ⁶⁰Co-γ-rays, and in white blood cells and bone marrow cells of X-irradiated mice. A dose-related DNA unwinding was observed and repair of DNA lesions was observed up to 60 min after irradiation.