Quantification of Photoproducts in Mammalian Cell DNA Using Radioimmunoassay

Radioimmunoassay (RIA) is a competitive binding assay between an unlabeled and a radiolabeled antigen for binding to antibody raised against that antigen. For the development of this technique, Yalow and Berson ( 1 ) received the Nobel Prize in Medicine. For detailed theory and troubleshooting of RIA, see Harlow and Lane ( 2 ) and Chard ( 3 ). We have adapted this technique to the measurement of specific DNA photoproducts in the DNA of UV-irradiated cells ( 4 , 5 ). The following description is given for quantification of cyclobutane pyrimidine dimers (CPDs) and pyriminidine(6-4)pyrimidinone photoproducts ([6-4]PPs) in DNA using RIA. For convenience, the radiolabeled antigen is referred to as the “probe,” and the unlabeled competitor as the “sample” or “standard.” The amount of radiolabeled antigen bound to antibody is determined by separating the antigen-antibody complex from free antigen by secondary antibody (Fig. 1 ). The amount of radioactivity in the antigen-antibody complex in the presence of known amounts of competitor (i.e., standards) can then be used to quantify the amount of unknown sample present in the reaction. The sensitivity of the RIA is determined by the affinity of the antibody and specific activity of the radiolabeled antigen. Using high-affinity antibody and probe labeled to a high specific activity, the reaction can be limited to such an extent that extremely low levels of damage in sample DNA can be detected. This particular procedure has resulted from 15–20 years of research and has proven to be a reliable and facile technique for measuring DNA damage and repair end points.   Fig. 1.  Diagram of RIA protocol. Top, Antibodies are raised against a specific type of DNA lesion, e.g., CPD. A variety of methods and treatments can be used to damage or modify bases in DNA for use as an immunogen. Middle: Binding activity of antisera is characterized. Bottom, RIA is used to measure lesion levels in sample DNA.