Ferritin-labeled rabbit Fab fragments for the single-step detection of benzo[a]pyrene-diol-epoxide adducts in DNA by electron microscopy.
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 method presents the following advantages: (i) it avoids cross-linking of separate adducts by the same IgG molecule; and (ii) it requires only one antigen-antibody reaction and a single purification step, allowing analysis of very small amounts of DNA.
Brylawski
B P
BP
Department of Pathology, University of North Carolina School of Medicine, Chapel Hill 27599-7525.
Cordeiro-Stone
M
M
Kaufman
D G
DG
D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; D000818:Animals; D000918:Antibody Specificity; D001564:Benzo(a)pyrene / Q000032:analysis*; D002417:Cattle; D004247:DNA / Q000032:analysis*; D018736:DNA Adducts; D004249:DNA Damage; D005293:Ferritins; D007140:Immunoglobulin Fab Fragments / Q000276:immunology; D066298:In Vitro Techniques; D008854:Microscopy, Electron / Q000379:methods
D018736:DNA Adducts; D007140:Immunoglobulin Fab Fragments; C056612:benzo(a)pyrene-DNA adduct; D001564:Benzo(a)pyrene; D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; D004247:DNA; D005293:Ferritins