Wild C P, Smart G, Saffhill R, Boyle J M
Carcinogenesis. 1983 Dec;4(12):1605-9. doi: 10.1093/carcin/4.12.1605.
Mouse monoclonal and rabbit polyclonal antibodies have been prepared against O6-methylguanosine complexed with bovine serum albumin. In radioimmunoassay 50% inhibition of binding of [3H]O6-methyldeoxyguanosine (O6-MedG) was obtained with 0.3 pmol or 0.16 pmol unlabelled O6-MedG using monoclonal or polyclonal antibodies having affinity constants of 3.0 X 10(9) and 5.9 X 10(9) I mol-1 respectively. Cross-reactivity with normal nucleosides and other adducts was determined. Sensitivity and reproducibility were improved by chromatographic separation using an Aminex A6 column eluted with 10 mM NH4HCO3 buffer which allowed quantitation of one molecule O6-MedG per 10(7) molecules dG in 2 mg calf thymine DNA. Values for O6-MedG from 14 samples of rat liver and kidney DNA analysed by the method described and by radiochromatography on Sephadex G10 were almost identical (correlation coefficient, 0.98). A modified procedure for the purification of 5-25 micrograms DNA from 1-5 X 10(6) fibroblasts applied to polycarbonate filters was used in providing further validation of the RIA system by measuring the persistence of O6-MedG in cell lines of known repair capacity.
已制备出针对与牛血清白蛋白复合的O6-甲基鸟苷的小鼠单克隆抗体和兔多克隆抗体。在放射免疫分析中，使用亲和常数分别为3.0×10⁹和5.9×10⁹ I mol⁻¹的单克隆抗体或多克隆抗体时，0.3 pmol或0.16 pmol未标记的O6-甲基脱氧鸟苷（O6-MedG）可使[³H]O6-甲基脱氧鸟苷（O6-MedG）的结合受到50%的抑制。测定了与正常核苷和其他加合物的交叉反应性。使用用10 mM NH₄HCO₃缓冲液洗脱的Aminex A6柱进行色谱分离，提高了灵敏度和重现性，该方法可对2 mg小牛胸腺DNA中每10⁷个dG分子中的一个O6-MedG分子进行定量。用所述方法和在Sephadex G10上进行放射色谱分析对14个大鼠肝脏和肾脏DNA样品中的O6-MedG值进行分析，结果几乎相同（相关系数为0.98）。采用一种改进的方法从1 - 5×10⁶个成纤维细胞中纯化5 - 25微克DNA，将其应用于聚碳酸酯滤膜，通过测量已知修复能力的细胞系中O6-MedG的持久性，进一步验证了放射免疫分析系统。
