Ferritin-labeled rabbit Fab fragments for the single-step detection of benzo[a]pyrene-diol-epoxide adducts in DNA by electron microscopy

Carcinogenesis
B P BrylawskiD G Kaufman

Abstract

In this paper we describe an immunological method for the visualization of (+/-)7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10- tetrahydrobenzo[a]pyrene (BPDE-I) adducts in DNA by electron microscopy (EM). The immunoglobulin fraction of rabbit antiserum specific for BPDE-I adducts was digested with papain, the Fab fragments were purified by affinity chromatography on protein A-Sepharose and cross-linked to ferritin. The reactivity of the Fab fragments coupled to ferritin was determined by using anti-ferritin antibodies to precipitate the complexes formed between ferritin-labeled Fab fragments and BPDE-I-modified DNA that had been uniformly labeled with [14C]thymidine. DNA from cells treated with BPDE-I in culture was reacted with ferritin-labeled Fab fragments, separated from unreacted Fab using a Sepharose CL-4B column, and examined by EM. An aliquot of the same DNA was used to determine the level of BPDE-I adduction using an enzyme-linked immunosorbent assay (ELISA). Close agreement was found between the levels of adduction determined by ELISA and EM. A good correlation was also found between the level of adduction measured by EM and scintillation spectrometry when DNA was modified with [3H]BPDE-I in vitro. The EM...Continue Reading

Citations

Oct 28, 2006·FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology·Paul D ChastainDavid Kaufman

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