High selectivity of polyclonal antibodies against DNA modified by diastereomeric benzo[c]phenanthrene-3,4-diol-1,2-epoxides.
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.
Butch
E R
ER
Department of Medicinal Chemistry, School of Pharmacy and Pharmacal Sciences, Purdue University, West Lafayette, IN 47907.
Lau
H H
HH
Shaw
K L
KL
Smolarek
T A
TA
Schmerold
I
I
Anderson
J N
JN
Baird
W M
WM
Yagi
H
H
Jerina
D M
DM
D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide / Q000032:analysis* / Q000737:chemistry; D000818:Animals; D000906:Antibodies; D000918:Antibody Specificity; D001667:Binding, Competitive; D004247:DNA / Q000032:analysis* / Q000737:chemistry; D018736:DNA Adducts; D004797:Enzyme-Linked Immunosorbent Assay; D010616:Phenanthrenes / Q000032:analysis* / Q000737:chemistry; D011817:Rabbits; D013237:Stereoisomerism
D000906:Antibodies; D018736:DNA Adducts; D010616:Phenanthrenes; C043931:benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide-DNA; C090203:1,2-epoxy-3,4-dihydroxy-1,2,3,4-tetrahydrobenzo(c)phenanthrene; D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; D004247:DNA