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Detection of DNA adducts of benzo[a]pyrene using immunoelectrophoresis with laser-induced fluorescence: Analysis of A549 cells by Woei G. Tan; Trevor J. Carnelley; Paula Murphy; Hailin Wang; Jane Lee; Sharon Barker; Michael Weinfeld; X.Chris Le is a Chemistry article available to read on EtoBox.

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Detection of benzo [a]pyrene diol epoxide (BPDE)-damaged DNA in a human lung carcinoma cell line (A549) has been performed using free zone affinity capillary electrophoresis with laser-induced fluorescence (LIF). Using BPDE as a model carcinogenic compound, the speed, sensitivity and specificity of this technique was demonstrated. Under free zone conditions, an antibody bound adduct was baseline-resolved from an unbound adduct in less than 2 min. The efficiencies of 5 6 separation were in excess of 6?10 and 1?10 plates per meter for the antibody-bound and unbound adducts, respectively. Separation using a low ionic strength buffer permitted the use of a high electric field (830 V/ cm) without the loss of 210 resolving power. Using LIF detection, a concentration detection limit of roughly 3?10 M was achieved for a 90-mer oligonuleotide containing a single BDPE. The use of formamide in the incubation buffer to enhance denaturing of DNA did not affect the stability of the complex between the antibody and the adducts. Using a fluorescently labeled BPDE-modified DNA adduct probe, a competitive assay was established to determine the levels of BPDE-DNA adducts in A549 cells.

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Author

Woei G. Tan; Trevor J. Carnelley; Paula Murphy; Hailin Wang; Jane Lee; Sharon Barker; Michael Weinfeld; X.Chris Le

Publisher

Elsevier Science; Elsevier ; Elsevier BV (ISSN 1873-3778)

Published

2001

Language

EN

Field

Chemistry (Physical Sciences)