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Detection of DNA adducts of benzo[a]pyrene using immunoelectrophoresis with laser-induced fluorescence. Analysis of A549 cells.

Authors :
Tan WG
Carnelley TJ
Murphy P
Wang H
Lee J
Barker S
Weinfeld M
Le XC
Source :
Journal of chromatography. A [J Chromatogr A] 2001 Jul 27; Vol. 924 (1-2), pp. 377-86.
Publication Year :
2001

Abstract

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 separation were in excess of 6 x 10(5) and 1 x 10(6) 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 resolving power. Using LIF detection, a concentration detection limit of roughly 3 x 10(-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.

Details

Language :
English
ISSN :
0021-9673
Volume :
924
Issue :
1-2
Database :
MEDLINE
Journal :
Journal of chromatography. A
Publication Type :
Academic Journal
Accession number :
11521887
Full Text :
https://doi.org/10.1016/s0021-9673(01)00987-6