Immunological detection of DNA damage caused by melphalan using monoclonal antibodies.
Immunological detection of DNA damage caused by melphalan using monoclonal antibodies.
复制标题
使用单克隆抗体对马法兰引起的 DNA 损伤进行免疫学检测。
作者：
M. Tilby;J. Styles;C. Dean
DNA上美法仑加合物的免疫学检测应允许新类型的临床和实验研究。五个克隆的大鼠杂交瘤细胞系的衍生，每一个产生的抗体，结合到DNA烷基化与美法仑（苯丙氨酸氮芥），但不正常的DNA。使用竞争性荧光酶联免疫吸附试验进一步表征一种美法仑特异性抗体（MP5/73）。使用变性DNA，每个测定孔30 fmol的总美法仑加合物（使用放射性美法仑测定）引起50%的抗体结合抑制。变性对照DNA在45微克（130 nmol）/孔（测试的最大浓度）下引起16至24%的抑制。RNA上的加合物的行为与变性DNA上的加合物相似。天然DNA上的加合物在272至1335 fmol/孔下引起50%的抑制，这取决于烷基化频率和超声处理。天然对照DNA在45微克/孔下未引起可检测的抑制。抗体识别的加合物是热不稳定和碱不稳定的。在75%甲酰胺存在下，通过适度加热使烷基化DNA变性，在50 fmol/孔下得到50%的抑制，表明只有5%的识别加合物可以结合天然DNA中的抗体。
Immunological detection of melphalan adducts on DNA should permit new types of clinical and experimental investigations. Five cloned rat hybridoma cell lines were derived, each producing an antibody that bound to DNA alkylated with melphalan (phenylalanine mustard) but not to normal DNA. Further characterization of one melphalan specific antibody (MP5/73) used a competitive fluorogenic enzyme-linked immunoabsorbent assay. Using denatured DNA, 50% inhibition of antibody binding was caused by 30 fmol of total melphalan adducts (determined using radioactive melphalan) per assay well. Denatured control DNA caused 16 to 24% inhibition at 45 micrograms (130 nmol)/well, the maximum concentration tested. Adducts on RNA behaved similarly to those on denatured DNA. Adducts on native DNA caused 50% inhibition at 272 to 1335 fmol/well dependent upon alkylation frequency and sonication treatment. Native control DNA caused no detectable inhibition at 45 micrograms/well. The adducts recognized by the antibody were thermo- and alkali labile. Denaturation of the alkylated DNA by moderate heating in the presence of 75% formamide gave 50% inhibition at 50 fmol/well, indicating that only 5% of the recognized adducts could bind antibody in native DNA.