A sensitive color ELISA for detecting polycyclic aromatic hydrocarbon-DNA adducts in human tissues

Abstract

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.