Design and Applications of Anti Albumin-Adduct Antibodies to Assess Human Exposure to Aromatic Hydrocarbons.

摘要

Aromatic hydrocarbons are ubiquitous pollutants. Many of the hydrocarbons such as benzene and benzo[a]pyrene are genotoxic and are known risk factors for cancers. The genotoxicity comes from the metabolic activation of the parent compound to a reactive electrophilic form, which binds to the nucleophilic DNA and albumin to form stable adducts. Because adduct represents the biologically effective dose and albumin has relatively long half-life (∼1 month) and is abundant in blood (∼45 mg/ml), albumin adduct becomes a promising candidate to assess exposure to aromatic hydrocarbons in cancer epidemiology. This dissertation can be roughly divided into two parts. In the first part, an antibody was used to quantitatively assess benzo[ a]pyrene exposure. A sandwich enzyme-linked immunosorbent assay (ELISA), using a commercially available antibody, was developed to measure benzo[a]pyrene diol epoxide - albumin adduct (BPDE-HSA). The assay is much simpler and faster to use and is 10 times more sensitive than the traditional competitive ELISA to detect BPDE-HSA. The method was further revised to be directly compatible with plasma, hence eliminated all the tedious HSA isolation and purification steps. Finally, the revised assay was validated with two different sets of archived plasma samples. In the second part, another antibody was used in an enrichment step for the albumin adductomics. To characterize albumin adducts with liquid chromatography - mass spectrometry, they have to be digested and detected as peptide adducts. Cysteine (Cys34) is a known nucleophilic hotspot on the albumin that scavenges reactive aromatic electrophiles in blood (such as benzoquinone and naphthoquinones), therefore a semi-targeting antibody was designed to capture all peptides with electrophiles bound on the Cys34 (T3 adducts). After incorporating the antibody enrichment step into the adductomic workflow, results shown that the antibody enrichment has huge potential to increase the detection rate of low abundant T3 adducts. The major significance from this study is that the innovations are applicable to the exposure to other reactive chemicals. With a suitable capture antibody, the sandwich ELISA can be used to assess other exposures such as aflatoxin (in form of aflatoxin albumin adduct), while the semi-targeting antibody can be tailored to capture albumin peptide with a different locus of exposure significant, such as lysine.