Several immunological methods have been developed during the course of the previous grant for the detection of carcinogen-modified DNA components. These studies have indicated that immunoassays are rapid, extremely sensitive and specific probes. 0(6)-EtdGuo adducts and Thymidine dimers in DNA were measured at 0(6)-EtdGuo/dGuo molar ratios of 2.2 x 10 to the 7 and UV doses of less than 1 J/m2 in extremely small in vivo treated DNA samples. We now propose to develop monoclonal antibodies against 0(6)-EtdGuo, 0(6)-EtdThy and 0(2)-EtdCyt exhibiting higher affinity for individual carcinogen modified alterations within single- or double-stranded DNA. Antigens that more closely resemble the carcinogen-DNA adduct will be designed and used in modivied in vivo and in vitro immunization protocols. The antibodies of appropriate specificity will be chosen by screening the hybridoma clones against a combination of antigens. Sensitive assays like ELISA, USERIA and tracer RIA will be employed to screen the monospecific antibodies allowing quantitation of individual alterations directly in unhydrolysed DNA samples. Detection of specific base alterations in organ and cell types will be performed with damage specific antibodies by indirect assays using immunofluorescent, immunostaining, flow cytometry and autoradiographic procedures. Cryostat sections of the biopsies of human skin removed at various times post-UV-irradiation will be used for the immunologic quantitation of the initial and persistent levels of environmentally relevant pyrimidine dimers as a function of cell type. Fate of various modified bases induced in the DNA of rat mammary gland, brain and liver (organs sensitive and resistant to ENU induced tumors) will be studied utilizing these immuno-techniques to investigate the mechanisms of tumor induction as a function of cell and organ type.
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