Tilby M J, Johnson C, Knox R J, Cordell J, Roberts J J, Dean C J
Experimental Unit, Institute of Cancer Research, Sutton, Surrey, England.
Cancer Res. 1991 Jan 1;51(1):123-9.
An assay that is based upon a monoclonal antibody (ICR4) is described that enables the quantitation of cisplatin-induced adducts on DNA down to 3 nmol Pt/g DNA (i.e., 1 Pt adduct/10(6) bases), the level necessary to produce toxic effects in cells in vitro and in vivo, using just a few micrograms of DNA. Detection is possible below this level (although probably not necessary for in vivo studies) but the cross-reactivity of unmodified DNA sequences complicates absolute quantitation of adducts. Therefore, it will be possible to investigate the distribution of clinically useful platinum drugs in patients undergoing chemotherapy. Rats of strain F344 appeared to be the best, among several tested, for the production of antibodies to modified DNA, and they were used for the production of hybridomas. Fifteen hybridomas which secreted antibodies that bound to DNA that was highly modified with cisplatin but not to normal DNA were obtained. One (ICR4) was chosen for further characterization because of its relatively strong binding to DNA modified to a moderate level with cisplatin. The characterization included the development of a sensitive competitive enzyme-linked immunoabsorbent assay and the use of DNA that had been reacted with cisplatin both in vitro and in vivo. The levels of platination of both types of DNA samples were determined by atomic absorbance spectroscopy. For DNA that had been exposed to cisplatin in vitro, 50% inhibition of antibody binding was caused by about 15 fmol of total DNA-bound Pt/assay well. At moderate levels of platination, heating of the DNA solution at 100 degrees C for 5 min increased its immunoreactivity such that 50% inhibition was caused by 2.5 fmol Pt adducts/well. Pt adducts on DNA extracted from cells that had been treated with cisplatin were less immunoreactive than DNA treated with cisplatin in vitro, but after heating the immunoreactivity increased such that 50% inhibition in the assay was caused by 2 fmol Pt adduct/well. This sensitivity was invariant over a wide range of levels of platinum adduct frequency. DNA adducts formed by the second generation anticancer drug carboplatin were recognized similarly to the adducts formed by cisplatin, but those formed by the clinically inactive trans-diamminedichloroplatinum(II) or chloro(diethylenetriamine)-platinum(II)-chloride were not significantly immunoreactive. Control DNA cross-reacted in the competitive assay but the immunoreactivity per mol base was 10(7) times lower than the immunoreactivity of cisplatin adducts.
本文描述了一种基于单克隆抗体（ICR4）的检测方法，该方法能够定量检测顺铂诱导的DNA加合物，低至3 nmol Pt/g DNA（即1个铂加合物/10⁶个碱基），这是在体外和体内细胞中产生毒性作用所需的水平，仅需使用几微克DNA。低于此水平也可以进行检测（尽管可能对体内研究并非必要），但未修饰DNA序列的交叉反应使加合物的绝对定量变得复杂。因此，将有可能研究临床上有用的铂类药物在接受化疗患者中的分布情况。在几种受试大鼠品系中，F344品系的大鼠似乎最适合产生针对修饰DNA的抗体，因此被用于制备杂交瘤。获得了15个分泌抗体的杂交瘤，这些抗体与经顺铂高度修饰的DNA结合，但不与正常DNA结合。由于其中一个抗体（ICR4）与经顺铂中度修饰的DNA具有相对较强的结合力，因此被选择进行进一步表征。表征包括开发一种灵敏的竞争性酶联免疫吸附测定法，并使用在体外和体内与顺铂反应的DNA。两种类型的DNA样品的铂化水平通过原子吸收光谱法测定。对于体外暴露于顺铂的DNA，每测定孔中约15 fmol的总DNA结合铂可导致抗体结合受到50%的抑制。在中度铂化水平下，将DNA溶液在100℃加热5分钟可增加其免疫反应性，使得每孔2.5 fmol铂加合物可导致50%的抑制。从用顺铂处理过的细胞中提取的DNA上的铂加合物的免疫反应性低于体外经顺铂处理的DNA，但加热后免疫反应性增加，使得测定中50%的抑制是由每孔2 fmol铂加合物引起的。这种灵敏度在广泛的铂加合物频率水平范围内是不变的。第二代抗癌药物卡铂形成的DNA加合物与顺铂形成的加合物的识别方式相似，但临床上无活性的反式二氯二氨铂（II）或氯（二亚乙基三胺）铂（II）氯化物形成的加合物没有明显的免疫反应性。对照DNA在竞争性测定中发生交叉反应，但每摩尔碱基的免疫反应性比顺铂加合物的免疫反应性低10⁷倍。
