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.

Use of a monoclonal antibody to detect DNA damage caused by the anticancer drug cis‐diamminedichloroplatinum (II) in vivo and in vitro

“…Furthermore, DNA adducts could also be detected from cells treated with 5 ,uM cisplatin (210 nmol cisplatin/g DNA or 1.4 adducts/104 bases). This value was -70 fold lower than that reported by Tilby et al [36].,Although we did not measure the low limit of MAb62-5 sensitivity, it is Table 2 Comparison of DNA repair measured by different methods in HeLa and the resistant HeLa-CPR cells probably similar to the previously reported antibody [36]. If MAb62-5 showed reduced sensitivity, it could be explained by the suggestion that an immunogen with a high level of D/N modification causes low efficiency in detection [37].…”

“…Therefore, MAb62-5 as well as cisplatin DRP distinguishes ciplatinum modification from UV modification of the same DNA molecule. The sensitive detection of cisplatin-DNA adducts in vivo using monoclonal antibody has also been demonstrated by others [36]. Assays with apparently reduced sensitivities at low DNA modification levels have been described for other types of DNA damage [4244].…”

Using an RNAi Signature Assay To Guide the Design of Three-Drug-Conjugated Nanoparticles with Validated Mechanisms, In Vivo Efficacy, and Low Toxicity

“…These data indicate that the CisPtXL and NP-CisPt are both more similar to the transcription/translation inhibitors than to the DNA cross-linkers. To support this observation, we used a CisPt-DNA adduct specific antibody 18 to analyze genomic DNA isolated from murine lymphoma cells after 8 h of free cisplatin versus CisPtXL treatment. Cisplatin-DNA adducts were only detected (Figure S7) as a result of cisplatin (CisPt) treatment, and not CisPtXL , further confirming that the latter is not acting as a DNA cross-linker.…”

Covalent binding of nitrogen mustards to the cysteine-34 residue in human serum albumin

“…It is believed that these compounds act through inter-and intra-strand crosslinking of guanine residues in DNA, which process inhibits cell proliferation (for a review see for example Lawley and Phillips 1996). Adduct formation of nitrogen mustards with DNA has been studied extensively over the last two decades, and methods for (sensitive) detection of these adducts have been developed (Tilby et al 1995;Thulin et al 1996;McCartney et al 2001).…”

“…Firstly, it is of clinical importance, since less drug is available for DNA alkylation after covalent binding to proteins. Secondly, studies of the genotoxic effect of these agents can be facilitated by methods for quantifying the extent to which these drugs form adducts (Tilby et al 1995). In this respect it is assumed that the degree of DNA alkylation is more or less proportional to the degree of protein alkylation.…”