Comparison of various immunochemical assays for the detection of ethylene oxide-DNA adducts with monoclonal antibodies against imidazole ring-opened N7-(2-hydroxyethyl)guanosine: application in a biological monitoring study

Abstract

Monoclonal antibodies have been developed for the analysis of the predominant lesion in DNA induced by ethylene oxide (EtOx), namely N7-(2-hydroxyethyl)guanine (N7-EtOHGua). Two monoclonal antibodies raised against imidazole ring-opened N7-(2-hydroxyethyl)guanine (RON7-EtOHGua), N7EO-E and N7EO-11, and the previously isolated antibody N7E-102 were characterized by competitive ELISA with various inhibitors. N7EO-E and N7EO-11 recognize 2-hydroxyethyl lesions better than ethyl or methyl lesions, while N7E-102 recognizes 2-hydroxyethyl and ethyl modifications equally well. All antibodies show a preference for imidazole ring-opened adducts, bind better to adducts in DNA compared to alkylated nucleosides or bases and bind 10(6)- to 3 x 10(6)-fold less well to unmodified DNA. The sensitivity of detection of RON7-EtOHGua in DNA and in the nuclei of cells in situ by antibody N7EO-E was investigated in several assays. The immunoslot blot assay was the most sensitive method (0.34 RON7-EtOHGua per 10(6) nucleotides was detectable), followed by competitive ELISA, direct ELISA and in situ detection by immunofluorescence microscopy. The immunoslot blot assay was used to analyse N7-EtOHGua levels in white blood cell DNA from individuals exposed to EtOx (2-5 p.p.m.) and of controls. This exposure did not result in a statistically significant increase in the N7-EtOHGua level.

The application of mass spectrometry in molecular dosimetry: Ethylene oxide as an example

“…The antibodies show more preference for imidazole-opened adducts and bound adducts in DNA than normal nucleosides or bases. The monoclonal antibodies also bind to adducts 1-3 Â 10 6 -fold better than unmodified bases in DNA (van Delft et al, 1994). This method has a detection limit of 1 pmol/mmol guanine in a very small amount of DNA sample (3 mg).…”

“…Primary monoclonal antibodies are usually raised toward the imidazole ring-opened N7-HEG (Young et al, 1990;van Delft et al, 1994;Ball et al, 2004). When bound to N7-HEG, the primary antibodies are detected by secondary antibodies linked covalently to an enzyme such as alkaline phosphatase or peroxidase.…”

“…This method has a detection limit of 1 pmol/mmol guanine in a very small amount of DNA sample (3 mg). The disadvantage of this method is its poor specificity because the monoclonal antibodies show extensive cross-reactivity toward N7-methylguanine and N7-ethylguanine (van Delft et al, 1994).…”

“…The contents of N7-HEG in blood cells and lymphocytes appeared within similar ranges (Table 4). N7-HEG in lymphocytes varies from 0.21 to 1.85 adducts/10 7 nucleotides with GC/ HRMS analysis (Wu et al, 1999b), and not detectable to 2.6 adducts/10 7 nucleotides with analysis by immunochemical assay (van Delft et al, 1994), and 0.37 to 0.66 adducts/10 7 nucleotides with 32 P-postlabeling assay Zhao & Hemminki, 2002). The levels in granulocytes ranged from 1.6 to 240 adducts/10 7 nucleotides with GC/ECNCI-MS analysis (Kao & Giese, 2005;Yong et al, 2007).…”

Optimizing immunoslot blot assays and application to low DNA adduct levels using an amplification approach

“…The immunoslot blot (ISB) assay was first developed by Nehls and coworkers in 1984 (1). Since then the assay has been applied to the detection of several DNA adducts formed both in vitro (2)(3)(4)(5) and in vivo (6)(7)(8)(9)(10)(11)(12) and the use of both monoclonal (13,14) and polyclonal (2) antibodies has been described.…”