Immunoaffinity purification combined with 32P-postlabelling 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 [3H]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 32P-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 mumol/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 micrograms of DNA is used.
Cooper
D P
DP
Cancer Research Campaign Department of Carcinogenesis, Paterson Institute for Cancer Research, Manchester, UK.
Griffin
K A
KA
Povey
A C
AC
D002855:Chromatography, Thin Layer; D004247:DNA / Q000737:chemistry*; D006147:Guanine / Q000031:analogs & derivatives* / Q000032:analysis; D006801:Humans; D010761:Phosphorus Radioisotopes
D010761:Phosphorus Radioisotopes; D006147:Guanine; D004247:DNA; C008449:O-(6)-methylguanine