Degan P, Shigenaga M K, Park E M, Alperin P E, Ames B N
Division of Biochemistry and Molecular Biology, University of California, Berkeley 94720.
Carcinogenesis. 1991 May;12(5):865-71. doi: 10.1093/carcin/12.5.865.
An immunoaffinity column is described that facilitates the analysis of oxidative DNA damage. DNA adducts excised from DNA are excreted in urine and can be assayed as a measure of DNA damage in individuals. Polyclonal antibodies that recognize 8-hydroxy-2'-deoxyguanosine (oh8dG), a biomarker of oxidative damage to DNA, have been produced and their binding properties characterized. The antibodies, raised in rabbits following immunization with protein carrier-hapten conjugates prepared by covalently linking periodate-treated 8-hydroxyguanosine (oh8G) to bovine serum albumin (BSA) or casein, bind oh8dG with high affinity and selectivity, as measured by a competitive radioimmunoassay (RIA). Antibodies obtained from the rabbits immunized with the casein conjugate exhibited a binding affinity for oh8dG of 6.9 x 10(8) M-1. Studies on the relative binding affinities of these polyclonal antibodies for oh8dG, unmodified nucleosides, or derivatives of guanine indicate that the antibodies are suitable for the preparation of immunoaffinity columns that permit us to rapidly isolate oh8dG and 8-hydroxyguanine (oh8Gua) from urine. The high selectivity of the antibodies for oh8dG and oh8G reduces the amount of urinary contaminants previously observed in samples prepared by solid phase extraction, thus greatly facilitating the isolation of these damage products from urine. The relative binding affinity of these antibodies for oh8Gua and 2'-deoxyguanosine were approximately 7.6 x 10(3) and 7.4 x 10(4) fold lower respectively, than the binding affinity for oh8dG. The antibody can be used to quantitate oh8dG in enzymatic hydrolyzates of DNA with values comparable to those obtained by HPLC with electrochemical detection (HPLC-EC).
本文描述了一种有助于分析氧化性DNA损伤的免疫亲和柱。从DNA中切除的DNA加合物会随尿液排出，可作为个体DNA损伤的一种检测指标。已制备出识别8-羟基-2'-脱氧鸟苷（oh8dG）（一种DNA氧化损伤的生物标志物）的多克隆抗体，并对其结合特性进行了表征。通过将经高碘酸盐处理的8-羟基鸟苷（oh8G）与牛血清白蛋白（BSA）或酪蛋白共价连接制备的蛋白质载体-半抗原缀合物免疫兔子后获得的抗体，通过竞争性放射免疫分析（RIA）测定，其对oh8dG具有高亲和力和选择性。用酪蛋白缀合物免疫的兔子获得的抗体对oh8dG的结合亲和力为6.9×10⁸ M⁻¹。对这些多克隆抗体与oh8dG、未修饰核苷或鸟嘌呤衍生物的相对结合亲和力的研究表明，这些抗体适用于制备免疫亲和柱，使我们能够从尿液中快速分离出oh8dG和8-羟基鸟嘌呤（oh8Gua）。抗体对oh8dG和oh8G的高选择性减少了先前在固相萃取制备的样品中观察到的尿液污染物量，从而极大地促进了这些损伤产物从尿液中的分离。这些抗体对oh8Gua和2'-脱氧鸟苷的相对结合亲和力分别比其对oh8dG的结合亲和力低约7.6×10³和7.4×10⁴倍。该抗体可用于定量DNA酶解产物中的oh8dG，其值与通过高效液相色谱-电化学检测（HPLC-EC）获得的值相当。
