Cooper D P, Griffin K A, Povey A C
Cancer Research Campaign Department of Carcinogenesis, Paterson Institute for Cancer Research, Manchester, UK.
Carcinogenesis. 1992 Mar;13(3):469-75. doi: 10.1093/carcin/13.3.469.
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.
使用三种不同的活化载体来固定α - O6 - 甲基脱氧鸟苷（O6 - MedG）单克隆抗体。基于溴化氰琼脂糖、Affigel Hz和高碘酸盐活化琼脂糖（PIAS）的亲和凝胶分别能够结合1.4、1.5和6.2 nmol [3H]O6 - MedG/mg固定化IgG，并且在所有情况下结合物质的回收率均超过95%。仅在使用溴化氰凝胶时，正常核苷出现了显著的非特异性结合。PIAS α - O6 - MedG亲和凝胶能够从消化的合成寡核苷酸和体外甲基化的小牛胸腺DNA中纯化O6 - MedG - 3'-单磷酸，以便随后通过32P后标记和二维薄层层析进行检测。该联合方法应用于三个人类样本，发现O6 - MedG水平分别为0.39、0.38和0.45 μmol/mol 2'-脱氧鸟苷。当使用100 μg DNA时，联合方法的最低检测限预计约为10(8)个正常2'-脱氧鸟苷中的1个O6 - MedG（即每人细胞约30个损伤）。
