Povey A C, Cooper D P
Department of Carcinogenesis, Paterson Institute for Cancer Research, Manchester, UK.
Carcinogenesis. 1995 Jul;16(7):1665-9. doi: 10.1093/carcin/16.7.1665.
In this study, a combined immunoaffinity purification/32P-postlabelling procedure has been used to quantify O6-methyldeoxyguanosine-3'-monophosphate (O6-MedGp) in human DNA. DNA digests are subjected to a two-stage immunopurification in which the acetone-eluted fraction from the first stage is reapplied to a second immunocolumn, and the O6-MedGp specifically eluted using O6-methylguanosine (O6-MerG). O6-MedGp is then 32P-postlabelled in the presence of deoxyinosine-3'-monophosphate (dIp) as internal standard, separated by two-dimensional TLC and levels of the adduct quantified using storage phosphor technology. The recovery of O6-MedGp at levels between 0.4 and 500 fmol was 61%. Analysis of human DNA samples indicated that < 1 fmol O6-methyldeoxy-guanosine-5'-monophosphate (O6-MepdG) could be detected with a high degree of precision (coefficient of variation < 12%) during a 2 h exposure to a storage phosphor screen. The assay was then applied to 25 human samples from three separate populations, one of which was exposed to methylating agent chemotherapy, for which O6-methyl-deoxyguanosine (O6-MedG) levels had already been quantified by HPLC/radioimmunoassay. The results indicated a high degree of correlation between the two assays (r = 0.99). O6-MedGp was detected in all the samples analysed with levels ranging from 0.026 to 23.2 mumol O6-MedGp/mol dG. The minimum amount of O6-MepdG detected was 0.2 fmol. As there was no detectable signal in the area to which O6-MepdG maps in negative control samples, a detection limit based upon the signal/noise ratio was impossible to quantify. However the limit of detection of the storage phosphor technology itself was estimated by quantifying a visually identifiable compound, which mapped to the same region. The amount of this compound was determined to be 32 +/- 27 amol (n = 5). If a similar amount of O6-MepdG was detected from 50 micrograms of DNA, and assuming that the labelling efficiency and recovery was similar to that found in this study, then this would correspond to an adduct level of approximately 3 nmol O6-MedGp/mol dG.
在本研究中，采用免疫亲和纯化与³²P后标记相结合的方法对人DNA中的O6-甲基脱氧鸟苷-3'-单磷酸（O6-MedGp）进行定量。DNA消化产物先进行两阶段免疫纯化，第一阶段用丙酮洗脱的部分再应用于第二个免疫柱，然后用O6-甲基鸟苷（O6-MerG）特异性洗脱O6-MedGp。接着，在脱氧肌苷-3'-单磷酸（dIp）作为内标的情况下对O6-MedGp进行³²P后标记，通过二维薄层层析分离，并用磷屏成像技术对加合物水平进行定量。O6-MedGp在0.4至500飞摩尔水平的回收率为61%。对人DNA样本的分析表明，在磷屏成像2小时期间，能以高精度（变异系数<12%）检测到<1飞摩尔的O6-甲基脱氧鸟苷-5'-单磷酸（O6-MepdG）。然后将该检测方法应用于来自三个不同人群的25份人类样本，其中一组接受了甲基化剂化疗，其O6-甲基脱氧鸟苷（O6-MedG）水平已通过高效液相色谱/放射免疫分析法进行了定量。结果表明两种检测方法具有高度相关性（r = 0.99）。在所有分析的样本中均检测到O6-MedGp，其水平范围为0.026至23.2微摩尔O6-MedGp/摩尔dG。检测到的O6-MepdG的最小量为0.2飞摩尔。由于在阴性对照样本中O6-MepdG映射的区域没有可检测到的信号，因此无法根据信噪比来量化检测限。然而，通过对一种视觉上可识别的化合物进行定量来估计磷屏成像技术本身的检测限，该化合物映射到相同区域。该化合物的量测定为32±27阿托摩尔（n = 5）。如果从50微克DNA中检测到类似量的O6-MepdG，并假设标记效率和回收率与本研究中发现的相似，那么这将对应于约3纳摩尔O6-MedGp/摩尔dG的加合物水平。
