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© 1991 Oxford University Press

research-article

Immunoaffinity isolation of urinary 8-hydroxy-2'-deoxygunosine and 8-hydroxyguanine and quantitation of 8-hydroxy-2'deoxyguanosine in DNA by polyclonal antibodies

Paolo Degan 1, Mark K. Shigenaga, Eun-Mi Park, Peter E. Alperin and Bruce N. Ames 2

Division of Biochemixtry and Molecular Biology 401 Barker Hall, University of California, Berkeley, CA 94720, USA

An immunoaffinity column is described that facilitates the analysis of oxidative DNA adducts excised from DNA are excreted in urine and can assayed as a measure of DNA damage in individuals. Polyclonal antibodies that recognize 8-hydoroxy-2'-deoxyguanosine (oh8dG), a biiomarker of oxidative damage to DNA, have been produced and their binding properties characterized. The antibodies, raised in rabbits following immunization with protein carrier-hapten conjugates prepared by covalently linking periodate-treated 8-hydroxyguanosine (oh8dG to bovine serum albumin (BSA) or casein, bind ohn8dG with high affinity and selectivity, as measured by a competitive radio-immunoassy (RIA). Antibodies oboitained from the rabbits immunized with the casein conjugate exhibited a binding affinity for oh8dG of 6.9 108 x M–1. Studies on the relative binding affinities of these polyclonal antibodies for oh8dG, unmodified nucleosides, or derivatives of guanine indicate that the antibodies are suitable for the preparation of immuno-affinity dolumns that permit us to rapidly isolate oh8dG and 8-hydroxyguanine (oh8dG) from urine. The high selectivity of the antibodies for oh8dG and oh8dG reduces the amount of urinary contaminants previsouly observed in samples prepared by solid phase extraction, thus greatly facilitating the isolation of these damage products from urine. The relative binding affinity of these antibodies for oh8dG. The antiboty can be used to quantitate oh8dG in enzymatic hydrolyzated of DNA with values comparable to those obtained by HPLC with electrochemicaldetection (HPLC-EC).


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