The isolation of monoclonal antibodies selected for the detection of imidazole ring-opened N7-ethylguanine in purified DNA and in cells in situ. Crossreaction with methyl, 2-hydroxethyl and sulphur mustard adducts

Abstract

Monoclonal antibodies have been obtained against imidazole ring-opened N7-ethylguanine (RON7-EtGua) in DNA. The antibodies were selected for good performance in the ELISA with either DNA or nucleated blood cells as immobilized antigen. Antibodies thus selected were studied for their suitability for the in situ detection of RON7-EtGua in the nuclei of cells by means of immunofluorescence microscopy (IFM). Two antibodies have been characterized in detail with respect to specificity and sensitivity. Competitive ELISA demonstrated that the antibodies recognize not only RON7-EtGua but also the corresponding methyl and 2-hydroxyethyl components, with efficiencies that vary with the chemical environment (base, nucleoside or DNA), the nature of the alkyl group and the antibody. They have a clear specificity for the ring-opened alkyl adducts and show an at least 100-fold stronger preference for such structures in DNA when compared to the free nucleoside adducts. Furthermore, they hardly bind to non-alkylated DNA, and do not bind to guanosine or N1- or O6-ethylguanosine. Analysis by DNA-ELISA showed that the binding preference of antibody N7E-026 for ring-opened alkyl adducts is methyl approximately ethyl greater than 2-hydroxyethyl much greater than sulphur mustard, while that of N7E-102 is 2-hydroxyethyl greater than ethyl greater than methyl approximately sulphur mustard. Analysis of RON7-EtGua in DNA with competitive ELISA, DNA-ELISA and IFM showed that in all cases the lowest detection limit can be reached with antibody N7E-026. Competitive ELISA was the most sensitive method, followed by DNA-ELISA and IFM, with detection limits of 2.2, 16 and 23 RON7-EtGua/10(6) nucleotides respectively. In the DNA-ELISA, 12 methyl adducts/10(6) nucleotides can be detected with N7E-026 and 11 2-hydroxyethyl adducts/10(6) nucleotides with N7E-102.

Development and Application of a Sensitive and Rapid Immunoassay for the Quantitation of N7-Methyldeoxyguanosine in DNA Samples

“…Several techniques have been applied to the detection and quantitation of N7-MedG in human DNA, e.g., 32 Ppostlabeling (PPL) (19)(20)(21), HPLC with fluorescence (22) or electrochemical (23) detection, and enzyme-linked immunoassay (24). The PPL-based assays possess the greatest sensitivity and are thus considered to be the most appropriate approach for quantitating N7-MedG, particularly when the amount of DNA is limited.…”

Accurate and Sensitive Quantitation of N7-Methyldeoxyguanosine-3‘-monophosphate by 32P-Postlabeling and Storage-Phosphor Imaging

“…The DNA was then ethanol precipitated and redissolved in 10 mM KH2PO4‚KOH, pH 7.0, to give a final concentration of 1-1.5 mg/mL. The N7-MeG content of the methylated DNA was determined using an established HPLC-ECD modified procedure (13,19) Detector, Leiden, The Netherlands) following HPLC using a Chromspher C-18 column (5 µm, 100 mm × 3 mm; Chrompack, Vlissingen, The Netherlands) eluted with 25 mM H3PO4‚KOH, pH 6.0, containing 4% (v/v) methanol (flow rate was 0.5 mL/ min; retention time of N7-MeG was 7.5 min). The recovery of N7-MeG ranged from 85% to 95%; the coefficient of variation of repeat analyses was <2.5%.…”

Determination of N7- and O6-methylguanine in Rat Liver DNA after Oral Exposure to Hydrazine by Use of Immunochemical and Electrochemical Detection Methods

“…The protocol of the competitive ELISA, described in detail by Van Delft et al (1991), was briefly as follows. Various dilutions, in duplicate, of the inhibiting antigen, e.g., unmodified and methylated DNA, were mixed with a fixed amount of the monoclonal antibody N7E-026 [specific for imidazole ring-open N7-MeGua and N7-ethylguanine (N7-EtGua) in DNA; Van Delft et al, 1991].…”

“…Various dilutions, in duplicate, of the inhibiting antigen, e.g., unmodified and methylated DNA, were mixed with a fixed amount of the monoclonal antibody N7E-026 [specific for imidazole ring-open N7-MeGua and N7-ethylguanine (N7-EtGua) in DNA; Van Delft et al, 1991]. After incubation for 45 min at 37°C, the mixture was transferred to microtiter plates coated with alkali-treated and heat-denaturated /V-methyl-/V-nitrosourea (MNU)-modified calf thymus DNA (0.062 /xg per well; one N7-MeGua per 710 nucleotides) and incubated for 1 hr at 37°C.…”