Immunological detection of DNA damage caused by melphalan using monoclonal antibodies.

Immunological detection of melphalan adducts on DNA should permit new types of clinical and experimental investigations. Five cloned rat hybridoma cell lines were derived, each producing an antibody that bound to DNA alkylated with melphalan (phenylalanine mustard) but not to normal DNA. Further characterization of one melphalan specific antibody (MP5/73) used a competitive fluorogenic enzyme-linked immunoabsorbent assay. Using denatured DNA, 50% inhibition of antibody binding was caused by 30 fmol of total melphalan adducts (determined using radioactive melphalan) per assay well. Denatured control DNA caused 16 to 24% inhibition at 45 micrograms (130 nmol)/well, the maximum concentration tested. Adducts on RNA behaved similarly to those on denatured DNA. Adducts on native DNA caused 50% inhibition at 272 to 1335 fmol/well dependent upon alkylation frequency and sonication treatment. Native control DNA caused no detectable inhibition at 45 micrograms/well. The adducts recognized by the antibody were thermo- and alkali labile. Denaturation of the alkylated DNA by moderate heating in the presence of 75% formamide gave 50% inhibition at 50 fmol/well, indicating that only 5% of the recognized adducts could bind antibody in native DNA.