Use of a monoclonal antibody to detect DNA damage caused by the anticancer drug cis-diamminedichloroplatinum (II) in vivo and in vitro.
A monoclonal antibody, MAb62-5, was prepared and used to detect DNA damage due to the anticancer drug cis-diamminedichloroplatinum (II) (or cisplatin). ELISA competition indicated that the binding of MAb62-5 to cisplatin-DNA was competitively inhibited (50% control) by 210 nM of cisplatin bound to DNA, cisplatin/nucleotide (D/N) = 0.2. Using a DNA mobility shift assay, MAb62-5 binding activity was inhibited by 50% by approximately 50-fold molar excess of cisplatin-DNA adducts (D/N = 0.08), whereas there was less than 5% inhibition by UV-DNA adducts or mock-treated DNA. In addition, MAb62-5 showed a similar affinity to the cisplatin-DNA adducts as compared to an endogenous cisplatin-damaged DNA recognition protein. Using ELISA with this antibody, we have demonstrated a 2-fold enhancement in excision repair of cisplatin-DNA adducts in resistant HeLa cells. This is supported by the measurement of repair-associated DNA strand breaks using alkaline elution and host cell reactivation of transfected plasmid DNA carrying cisplatin damage. These findings also provide explanation for the complexicity of immunoassay in cells.
Department of Biochemistry, Chang Gung Medical College, Taoyuan, Taiwan, ROC.
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Chao
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Shieh
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Huang
D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D001667:Binding, Competitive; D002945:Cisplatin / Q000032:analysis* / Q000276:immunology / Q000378:metabolism / Q000494:pharmacology*; D018736:DNA Adducts / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D004249:DNA Damage; D004260:DNA Repair; D006367:HeLa Cells; D006801:Humans
D000911:Antibodies, Monoclonal; D018736:DNA Adducts; C057270:cisplatin-DNA adduct; D002945:Cisplatin