The use of rabbit polyclonal antibodies for the isolation of carcinogen-adducted DNA by immunoprecipitation

✍ Scribed by Bruna P. Brylawski; Marila Cordeiro-Stone; David G. Kaufman

• Publisher: John Wiley and Sons
• Year: 1991
• Tongue: English
• Weight: 599 KB
• Volume: 4
• Category: Article
• ISSN: 0899-1987
• DOI: 10.1002/mc.2940040410

✦ Synopsis

Polyclonal rabbit antibodies elicited against DNA with high levels of (+/-) 7r,8t-dihydroxy-9t,10t-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE-I) adducts were used to isolate DNA fragments modified by this carcinogen. DNA treated in vitro with different concentrations of BPDE-I was used as substrate in double-antibody immunoprecipitation reactions. The IgG fraction from immune rabbit serum (primary antibody) was reacted with single-stranded plasmid DNA bearing BPDE-I adducts, and the complexes were immunoprecipitated using goat antirabbit-IgG as secondary antibody. DNA was isolated from the immunoprecipitated pellet, blotted onto nitrocellulose or nylon, and hybridized with 32P-labeled sequences homologous to a fragment of the plasmid DNA used in the assay. The recovery of both DNA and adducts in the immunoprecipitated pellet increased with the level of carcinogen adduction of the DNA. The immunoprecipitation reaction appeared to be more efficient for fragments of DNA containing a high number of adducts. The amount of 32P-hybridizing material recovered by immunoprecipitation was virtually identical to the amount added to the reaction in DNA samples that contained three adducts per 10(3) nucleotides.