Gomes M, Santella R M
Cancer Center, School of Public Health, Columbia University, New York, New York 10032.
Chem Res Toxicol. 1990 Jul-Aug;3(4):307-10. doi: 10.1021/tx00016a006.
Two monoclonal antibodies (10C10 and 4D5) have been developed from the spleen cells of Balb/c mice immunized with 6-aminobenzo[a]pyrene covalently coupled to bovine serum albumin. These antibodies have been used in an immunoassay for the detection of benzo[a]pyrene and its metabolites in mouse urine. The antibodies were characterized in terms of sensitivity and specificity by competitive enzyme-linked immunosorbent assay (ELISA). With both antibodies, 50% inhibition of antibody binding is at 4 pmol of BP. The antibodies also cross-react with a number of BP metabolites as well as with several other polycyclic aromatic hydrocarbons (PAHs) including pyrene, 1-aminopyrene, and 7,12-dimethylbenz[a]anthracene but with different sensitivities. These results suggest that this assay will detect multiple PAH metabolites in urine. To test the assay on biological samples, mice were treated with [3H]BP, and urine was collected and digested with beta-glucuronidase and aryl sulfatase. Several methods were used to isolate BP and its metabolites from the urine, including ethyl acetate extraction, Sep-pak C18 cartridge chromatography, XAD2 resin chromatography, and immunoaffinity chromatography with antibody 4D5. Analysis of the urine extracts with antibody 4D5 gave 50% inhibition at 12-15 pmol of metabolites. Thus, quantitation of metabolites in this sample by competitive ELISA against a standard curve of BP would have underestimated actual metabolite levels by about 70%. This assay will be applied to the analysis of urines from individuals with environmental or occupational exposure. Since humans are usually exposed to BP in complex mixtures of PAHs, multiple metabolites may be present in the urine, making absolute quantitation difficult. This assay should thus serve as a general indicator of exposure to this class of chemicals.
用共价偶联牛血清白蛋白的6-氨基苯并[a]芘免疫Balb/c小鼠，从其脾细胞中制备了两种单克隆抗体（10C10和4D5）。这些抗体已用于免疫测定，以检测小鼠尿液中的苯并[a]芘及其代谢物。通过竞争酶联免疫吸附测定（ELISA）对抗体的敏感性和特异性进行了表征。两种抗体在4 pmol苯并[a]芘时均有50%的抗体结合抑制率。这些抗体还与多种苯并[a]芘代谢物以及其他几种多环芳烃（PAH）发生交叉反应，包括芘、1-氨基芘和7,12-二甲基苯并[a]蒽，但敏感性不同。这些结果表明该测定法可检测尿液中的多种PAH代谢物。为了在生物样品上测试该测定法，用[3H]苯并[a]芘处理小鼠，收集尿液并用β-葡萄糖醛酸酶和芳基硫酸酯酶消化。使用了几种方法从尿液中分离苯并[a]芘及其代谢物，包括乙酸乙酯萃取、Sep-pak C18柱色谱、XAD2树脂色谱以及用抗体4D5进行免疫亲和色谱。用抗体4D5分析尿液提取物时，在12 - 15 pmol代谢物时有50%的抑制率。因此，通过针对苯并[a]芘标准曲线的竞争ELISA对该样品中的代谢物进行定量，会使实际代谢物水平低估约70%。该测定法将应用于对环境或职业暴露个体的尿液分析。由于人类通常在PAH的复杂混合物中接触苯并[a]芘，尿液中可能存在多种代谢物，使得绝对定量变得困难。因此，该测定法应作为接触这类化学物质的一般指标。
