Immunoassays using capillary electrophoresis laser induced fluorescence detection for DNA adducts

摘要

Human DNA is exposed to a variety of endogenous and environmental agents that may induce a wide range of damage.The critical role of DNA damage in cancer development makes it essental to develop highly sensitive and specific assays for DNA lesions.We describe here ultrasensitive assays for DNA damage,which incorporate immuno-affinity with capillary electrophoresis (CE) separation and laser induced fluorescence (LIF) detection.Both competitive and non-competitive assays using CE/LIF were developed for the determination of DNA adducts of benzo[a]pyrene diol expoxide (BPDE).A fluorescently labeled oligonucleotide containing a single BPDE adduct was synthesized and used as a fluorescent probe for competitive assay.Binding between this synthetic oligonucleotide and a monoclonal antibody (Mab) showed both 1:1 and 1:2 complexes between the MAb and the oligonucleotide.The 1:1 and 1:2 complexes were separated by CE and detected with LIF,revealing binding stoichiometry information consistent with bidentate nature of the immunoglobulin G antibody.For non-competitive assay,a fluorescently labeled secondary antibody fragment F(ab')_2 was used as an affinity probe to recognize a primary antibody that was specific for the BDPE-DNA adducts,The ternary complex of BPDE-DNA adducts with the bound antibodies was separated from the unbound antibodies using CE detected with LIF for quantitation of the DNA adducts.The assay was used for the determination of trace levels of BPDE-DNA adducts in human cells.Analysis of cellular DNA from A549 human lung carcinoma cells that were incubated with low doses of BPDE (32 nM-1 muM) showed a clear dose-response relationship.BPDE is a potent environmental carcinogen,and the ultrasensitive assays for BPDE-DNA adducts are potentially useful for monitoring human exposure to this carcinogen and for studying cellular repair of DNA damage.