Development and validation of a new, sensitive immunochemical assay for O⁶-methylguanine in DNA and its application
Panagiotis Georgiadis1, Stella Kaila, Paraskevi Makedonopoulou
1National Hellenic Research Foundation, Institute Of Biological Research and Biotechnology, 48 Vas. Constantinou Avenue, Athens 11635, Greece. panosg@eie.gr
Background:
Investigations of the presence of the precarcinogenic DNA adduct O⁶-methylguanine (O6-meG) in humans and its association with exposure or cancer risk have been hindered by the absence of analytic methods of adequate sensitivity and throughput. We report the development, validation, and application of an ELISA-type assay for O6-meG appropriate for large-scale population studies.
Methods:
In the new analytic method, restriction enzymes are used to digest DNA to fragments of size expected to contain no more than one O6-meG residue. Anti-adduct antisera are used to transfer O6-meG-containing fragments to a solid surface, where they are detected using anti-ssDNA antisera, the high ratio of normal nucleotides to adducts providing a strong signal enhancement.
Results:
An assay with a limit of detection of 1.5 adducts/10⁹ nucleotides using 10 μg of DNA, a dynamic range of approximately two orders of magnitude and satisfactory precision and accuracy characteristics was established and validated. Analysis of samples from 120 subjects from the Rhea mother-child cohort in Crete led to the detection of O6-meG in 70% of maternal and 50% of cord blood buffy coat samples at mean levels of 0.65 and 0.38 adducts/10⁸ nucleotides, respectively.
Conclusions:
The frequent observation of O6-meG in human DNA is compatible with dietary compounds (e.g. N-nitroso compounds or their precursors), or endogenous processes being responsible for the formation of this adduct.
Impact:
The new assay opens the way for large-scale population studies of O6-meG as a biomarker of exposure or risk. The approach used in this assay can, in principle, be extended to any DNA adduct for which suitable antisera are available.
