A sensitive color ELISA for detecting polycyclic aromatic hydrocarbon-DNA adducts in human tissues
A sensitive color ELISA for detecting polycyclic aromatic hydrocarbon-DNA adducts in human tissues
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DOI：
10.1016/s0165-1161(96)90264-2
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发表时间：
1996-04-04
期刊：
影响因子：
--
通讯作者：
Santella, RM
中科院分区：
文献类型：
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作者：
Mumford, JL;Williams, K;Santella, RM
通过测定人体组织中的DNA加合物，采用抗血清识别苯并[a]芘二醇修饰的DNA的竞争性酶联免疫吸附试验（ELISA），（BPDE-1-DNA）和颜色或荧光终点检测已广泛用于定量PAH-DNA加合物，先前报道荧光ELISA（检测极限1加合物/10（8）个核苷酸）比用于测量PAH加合物的颜色ELISA（1/10（7））更灵敏（Santella等（1988）Carcinogenesis，9，1265-1269）。然而，与颜色测定相比，荧光测定具有重复测定之间的较大变化和较高背景水平的缺点。用新研制的抗BPDE-Ⅰ-DNA的抗血清，对显色ELISA进行了改进，使其具有与荧光ELISA相同的灵敏度，而所需样品量仅为荧光ELISA的33%。采用半孔平底威尔斯微量滴定板，优化了检测条件，并将改进后的显色ELISA方法应用于人尸检组织（包括心、肺、肝、肾、脾、胰和胃）DNA的检测。除脾和胃外，吸烟者的所有组织都显示出较高的PAH-DNA加合物（范围为0.3 - 19.0个加合物/10（7）个核苷酸），而非吸烟者的组织（0.3 - 3.7个加合物/10（7）个核苷酸）在两个单独的实验中，吸烟者的组织中，心脏显示出最高水平的DNA加合物。这项研究表明，具有高灵敏度的稳定颜色ELISA可以用于评估人类暴露于PAH。
Human exposure to polycyclic aromatic hydrocarbons (PAHs) has been determined by measurement of DNA adducts in human tissues, Competitive enzyme-linked immunosorbent assays (ELISAs) using antisera recognizing benzo[a]pyrenediol-epoxide-modified DNA (BPDE-I-DNA) and color or fluorescence endpoint detection have been used extensively for quantifying PAH-DNA adducts, The fluorescence ELISA (limit of detection 1 adduct/10(8) nucleotides) was previously reported to be more sensitive than the color ELISA (1/10(7)) for measuring PAH adducts (Santella et al. (1988) Carcinogenesis, 9, 1265-1269). However, the fluorescence assay has the disadvantages of greater variation among the replicates and higher background levels than the color assay. Using a newly developed antiserum against BPDE-I-DNA, we have modified the color ELISA so that it has the same sensitivity as the fluorescence ELISA and requires only 33% of the sample quantity needed for the fluorescence ELISA. The modifications included preincubation of the antiserum with the samples, using microtiter plates with half-size, flat bottom wells, and optimizing the assay conditions, The improved color ELISA was used to analyze DNA samples from human autopsy tissues, including heart, lung, Liver, kidney, spleen, pancreas and stomach from smokers and nonsmokers. With the exception of spleen and stomach, all tissues from smokers showed higher PAH-DNA adducts (ranging from 0.3 to 19.0 adducts/10(7) nucleotides) than the tissues from the nonsmokers (0.3 to 3.7 adducts/10(7) nucleotides) in two separate experiments, Among the tissues from smokers, heart showed the highest level of DNA adducts. This study demonstrates that a stable color ELISA with high sensitivity can be useful in assessing human exposure to PAH.