High selectivity of polyclonal antibodies against DNA modified by diastereomeric benzo[a]phenanthrene-3,4-diol-1,2-epoxides

Abstract

Polyclonal antibodies were developed in New Zealand White rabbits against DNA modified with diastereomeric benzo[c]phenanthrene-3,4-diol-1,2-epoxide (B[c]PhDE)-1 (4-hydroxyl and epoxide cis) and B[c]PhDE-2 (4-hydroxyl and epoxide trans). Antiserum developed against B[c]PhDE-2-DNA was stereoselective. In competitive ELISA assays using wells coated with 160 fmol B[c]PhDE-2-DNA adducts, B[c]PhDE-2-DNA gave 50% inhibition at 200 fmol adducts/well. B[c]PhDE-1-DNA required a 10-fold higher amount of adducts/well to give 50% inhibition. Benzo[a]pyrene-7,8-diol-9,10-epoxide-2-DNA and 7,12-dimethylbenz[a]anthracene-3,4-diol-1,2-epoxide-1-DNA caused only a 30% inhibition even at the highest doses tested (greater than 4000 fmol adducts/well). For antiserum developed against B[c]PhDE-1-DNA, 50% inhibition required 570 fmol B[c]PhDE-1-DNA adducts in wells coated with 100 fmol B[c]PhDE-1-DNA adducts. 7,12-Dimethylbenz[a]anthracene-3,4-diol-1,2-epoxide-1-DNA and B[c]PhDE-2-DNA were also effective competitors: they caused 50% inhibition at 1900 and 1800 fmol adducts/well respectively. In contrast, benzo[a]pyrene-7,8-diol-9,10-epoxide-2-DNA gave no inhibition at the highest dose of competitor tested (4050 fmol adducts/well). Antisera from three rabbits immunized with B[c]PhDE-2-DNA demonstrated similar antigen specificities. The properties of these antisera differ from those reported previously for antibodies developed against benzo[a]pyrene-DNA in that they show selectivity for DNA modified by specific hydrocarbon diolepoxides, in one case for B[c]PhDE-2-DNA and in the other for B[c]PhDE-DNA or 7,12-dimethylbenz[a]anthracene-3,4-diol-1,2-epoxide-1-DNA. The specificity of these antisera will facilitate analysis of the modification of DNA by different polycyclic aromatic hydrocarbon diolepoxides.

Development of a Monoclonal Antibody Recognizing Benzo[c]phenanthrenediol Epoxide-DNA Adducts: Application to Immunohistochemical Detection of DNA Damage

“…Previously, a polyclonal antiserum was developed against B[c]PhDE-DNA (18). While monoclonal antibody 10F9 is slightly more sensitive than the polyclonal antiserum (50% inhibition at 200 fmol of B[c]PhDE-DNA adduct), its major advantage is that large amounts can be produced making it more readily available.…”

“…Several polyclonal antisera and monoclonal antibodies recognizing BP diol epoxide-modified DNA have been developed (15)(16)(17) and used in enzyme-linked immunosorbent assays (ELISA) to detect adducts produced in vitro and in blood and tissue samples of humans with occupational, lifestyle, dietary, and environmental exposure (reviewed in ref 13). More recently, a polyclonal antiserum to B[c]-PhDE-DNA was also developed and characterized (18). Here, we report the development of a monoclonal antibody specific for B[c]PhDE-DNA and a sensitive ELISA for quantitation of DNA adduct levels.…”

Selective Recognition of Substituted Chrysene-Diol Epoxide-2-DNA Adducts by Antiserum prepared Against DNA Adducts of Benzo[c]Phenanthrene-Diol Epoxide-2

“…In contrast, B[c]PhDE-2 is a potent tumorigen in mouse skin, newborn mice and rat mammary glands [7,8,91 and is also mutagenic in bacteria and mammalian cells [lo]. These antisera have previously been found to be highly stereoselective for B[c]PhDE-2-DNA adducts having a 10-fold lower affinity for B[c]PhDE-1 -DNA adducts (epoxide oxygen and 4-hydroxyl group cis) as determined by competitive ELISAs [3]. [dG-dC] to similar extents, suggesting that the DNA base modified was not an important factor for antisera recognition [3].…”

“…These antisera have previously been found to be highly stereoselective for B[c]PhDE-2-DNA adducts having a 10-fold lower affinity for B[c]PhDE-1 -DNA adducts (epoxide oxygen and 4-hydroxyl group cis) as determined by competitive ELISAs [3]. [dG-dC] to similar extents, suggesting that the DNA base modified was not an important factor for antisera recognition [3]. The difference in the ability of the antisera to recognize DNA modified with B[a]PDE-2, which binds extensively with deoxyguanosine (dG) and 5,6-diMeCDE-2, which binds with deoxyadenosine (dA) as well as dG, was probably due to the structure of the PAHDE adduct rather than to the base modified by the PAHDE [I, 12-141. To determine which structural features of PAHDE would influence recognition of their adducts by B[c]PhDE-2-DNA antisera, competitive ELISAs were performed utilizing DNA that was modified with several structurally related PAHDE-2 isomers.…”

“…Fig. 1) were synthesized as described previously [13,17,18,19,20,211 aqueous solution and were extracted to remove unreacted tetrols [3]. The extents of DNA modification for the PAHDE-DNA were determined by nuclease Pl/prostatic acid phosphatase digestion, [T-~~PIATP post-labeling and reversed-phase HPLC analysis as described previously [22].…”