A monoclonal antibody that recognizes alkali-stabilized melphalan-DNA adducts and its application in immunofluorescence microscopy.

Abstract

Monoclonal antibodies were produced that recognized alkali-stabilized modifications of DNA formed by the anticancer drug melphalan in order to permit measurement of melphalan-DNA adducts in individual cells by immunofluorescent staining. Antibody Amp4/42 did not bind to alkali-treated control DNA or to DNA that had been alkylated with melphalan but not exposed to alkali. In a competitive enzyme-linked immunoadsorbent assay using DNA that had been reacted with radioactive melphalan in simple solution a 50% reduction in assay signal was caused by approximately 100 fmol total melphalan-DNA adducts/assay well. This sensitivity was only slightly influenced by heat denaturation of the DNA before alkylation or by the frequency of alkylated sites on DNA. The heat stability of the adducts recognized by Amp4/42 was greatly increased by the alkali-induced change which, in 0.1 M NaOH at 37 degrees C, was complete by 30 min. Amp4/42 appears to recognize a ring-opened structure resulting from alkaline hydrolysis of 7-alkyldeoxyguanosine. Melphalan-DNA adducts formed in mammalian cells showed an alkali-induced increase in immunoreactivity which occurred at a similar rate to that seen in DNA that had been alkylated in simple solution, but their maximum overall immunoreactivity was approximately 10-fold lower. This indicated that in cells the adducts recognized by Amp4/42 were formed or persisted as a smaller proportion of total adducts compared with alkylation of pre-purified DNA in simple solution. This antibody permitted immunofluorescent detection of melphalan-DNA adducts in single cells.