Immunoassay of O6-methyldeoxyguanosine in DNA: The use of polyethylene glycol to separate bound and free nucleoside

Abstract

Radioimmunoassay (RIA) method has been established for O 6-methyldeoxyguanosine ( O 6-medGuo), using precipitation with polyethylene glycol (PEG) to separate bound and free alkyldeoxyguanosine and antisera of higher affinity (1 × 10 9 M −1) and titre (1:10000) than previously reported. The lower limit of detection (2 S.D. from zero standard) is 40 pg (0.14 pmol) of O 6-medGuo. Hydrolysates of non-alkylated DNA extracts show interference in the precipitation of antigen-antibody complex, thus making necessary the construction of a standard curve with the same amount of DNA hydrolysates as in the sample to be analysed. Precision of the method was assessed using DNA hydrolysates from livers of rats injected with 15 mg/kg dimethylnitrosamine (DMN). Within assay variation of a single DNA extract was 85.9 ± 5.9 ng/mg DNA ( n = 5, CV = 6.9%). Day-to-day variation of the assay for four DNA extracts measured over 3 days were (ng/mg DNA S.D.), 99.8 ± 6.9, CV = 6.9%; 88.2 ± 6.5, CV = 7.4%; 81.3 ± 3.9, CV = 4.8%; 77.0 ± 6.4 CV = 8.3%. Coefficient of variation was 10.8% with DNA hydrolysate spiked with a known concentration of O 6-medGuo and assayed ( n = 5). Cross-reactivity of one of our best antisera coded R68/4, and raised against O 6-medGuo-BSA conjugate were: O 6-methylguanosine ( O 6-meGuo), 9.6%; O 6-methylguanine ( O 6-meGua), 0.4%; O 6-ethylguanine ( O 6-EtGua), 0.12%; 7-methylguanosine (7-meGuo), 0.03%; 2′-deoxyadenosine (2′-dAdo), 0.007%; 2′-deoxyguanosine-5′-monophosphoric acid (2′-dGuoMP), 2′-deoxyguanosine (2′-dGuo), 7-meGua and 7-meGuo did not significantly inhibit the binding of O 6-me[1′-2′- 3H]dGuo at concentrations of up to 10 μg/tube. The application of such methods underline the potential of RIAs for quantification of DNA components structurally modified by non-radioactive carcinogens.