IMMUNOAFFINITY PURIFICATION COMBINED WITH P-32 POSTLABELING FOR THE DETECTION OF O6-METHYLGUANINE IN DNA FROM HUMAN TISSUES

Three different activated supports were used to immobilize alpha-O6-methyldeoxyguanosine (O6-MedG) monoclonal antibody. Affinity gels based on CNBr Sepharose, Affigel Hz and periodate-activated Sepharose (PIAS) were able to bind 1.4, 1.5 and 6.2 nmol [H-3]O6-MedG/mg immobilized IgG respectively, and recovery of bound material exceeded 95% in all cases. Significant non-specific binding of normal nucleosides occurred only when using the CNBr gel. PIAS alpha-O6-MedG affinity gel was able to purify O6-MedG-3'-monophosphate from digested synthetic oligonucleotides and in vitro-methylated calf thymus DNA to allow subsequent detection by P-32-postlabelling and two-dimensional TLC. The combined method was applied to three human samples and O6-MedG levels of 0.39, 0.38 and 0.45-mu-mol/mol 2'-deoxyguanosine were found. The minimum detection limit of the combined method is expected to be approximately 1 O6-MedG in 10(8) normal 2'-deoxyguanosines (i.e. approximately 30 lesions per human cell) when 100-mu-g of DNA is used.