Casale G P, Rogan E G, Stack D, Devanesan P, Cavalieri E L
Eppley Institute for Research in Cancer, University of Nebraska Medical Center, Omaha 68198-6805, USA.
Chem Res Toxicol. 1996 Sep;9(6):1037-43. doi: 10.1021/tx950203e.
Molecular dosimetry of depurinating DNA adducts of benzo[alpha]pyrene (BP) is a promising new approach to measurement of cancer risk associated with exposure to polycyclic aromatic hydrocarbons (PAH). Depurinating adducts of BP are spontaneously released from DNA and can be detected in urine. As a first step toward developing a monoclonal antibody (MAb)-based molecular dosimetry for depurinating DNA adducts of BP, a MAb (MAb CB53) has been produced with high specific affinity for 7-(benzo[alpha]pyren-6-yl)guanine (BP-6-N7Gua), a major depurinating adduct of BP. Production of this MAb was dependent on the successful synthesis of an effective immunogen consisting of the hydrophobic BP-6-N7Gua coupled to carrier protein via a rigid spacer arm. A competitive enzyme-linked immunosorbent assay (ELISA) for BP-6-N7Gua has been developed with MAb CB53 and has been applied to evaluation of MAb binding and to quantitation of BP-6-N7Gua in a biological sample. The MAb binds with high affinity to BP-6-N7Gua (Ka = 1.4 x 10(8) M-1) and to BP-6-N7Ade (Ka = 0.7 x 10(8) M-1), another major depurinating DNA adduct of BP, but discriminates well between BP and BP-6-N7Gua. BP-6-N7Gua produces 50% inhibition at 750 fmol in the competitive ELISA, whereas BP produces 50% inhibition at 960 000 fmol. Binding affinities to selected PAH, BP-DNA adducts, and BP metabolites indicate significant contributions of the hydrophobic region C-3, C-4, and C-5 of BP and the polar oxygen of guanine to MAb/adduct binding. In a preliminary test of the utility of the competitive ELISA for quantitation of BP-6-N7Gua in urine samples, the assay (sensitivity: 200 fmol per well) produced an accurate determination of the adduct added to normal human urine.
苯并[a]芘（BP）脱嘌呤DNA加合物的分子剂量测定是一种很有前景的新方法，用于测量与多环芳烃（PAH）暴露相关的癌症风险。BP的脱嘌呤加合物会从DNA中自发释放，并可在尿液中检测到。作为开发基于单克隆抗体（MAb）的BP脱嘌呤DNA加合物分子剂量测定法的第一步，已制备出对7-（苯并[a]芘-6-基）鸟嘌呤（BP-6-N7Gua）具有高特异性亲和力的单克隆抗体（MAb CB53），BP-6-N7Gua是BP的一种主要脱嘌呤加合物。该单克隆抗体的产生依赖于一种有效免疫原的成功合成，该免疫原由通过刚性间隔臂与载体蛋白偶联的疏水性BP-6-N7Gua组成。已利用MAb CB53开发了一种针对BP-6-N7Gua的竞争性酶联免疫吸附测定（ELISA），并已将其应用于评估单克隆抗体的结合以及定量生物样品中的BP-6-N7Gua。该单克隆抗体与BP-6-N7Gua（Ka = 1.4 x 10^8 M^-1）和BP-6-N7Ade（Ka = 0.7 x 10^8 M^-1）具有高亲和力，BP-6-N7Ade是BP的另一种主要脱嘌呤DNA加合物，但能很好地区分BP和BP-6-N7Gua。在竞争性ELISA中，BP-6-N7Gua在750 fmol时产生50%的抑制，而BP在960000 fmol时产生50%的抑制。对选定的多环芳烃、BP-DNA加合物和BP代谢物的结合亲和力表明，BP的疏水区域C-3、C-4和C-5以及鸟嘌呤的极性氧对单克隆抗体/加合物结合有重要贡献。在对竞争性ELISA用于定量尿液样品中BP-6-N7Gua效用的初步测试中，该测定法（灵敏度：每孔200 fmol）准确测定了添加到正常人尿液中的加合物。
