6,4,4'-trimethylangelicin photoadduct formation in DNA: production and characterization of a specific monoclonal antibody.
The photochemical reactions of 6,4,4'-trimethylangelicin (TMA) with calf thymus DNA and an octanucleotide containing a single thymine have been characterized. HPLC analyses of enzymatically hydrolyzed TMA-DNA showed that isomeric forms of 4',5'-furan-side monoadducts were the major products. To develop monoclonal antibodies Balb c mice were immunized with the TMA-DNA complexed with methylated bovine serum albumin. The resultant antibodies were characterized by enzyme-linked immunosorbent assays (ELISA). The most sensitive antibody (7E3) has high specificity for TMA-DNA, very low cross-reactivity with DNA modified with either 4',5'-dimethylangelicin or 4'-methylangelicin and no cross-reactivity with non-modified DNA or with DNA modified with either 4'-aminomethyl-4,5',8-trimethylpsoralen or 8-methoxypsoralen. To characterize further this antibody, oligonucleotides containing specific TMA photoadducts were isolated from the photoreaction mixture by polyacrylamide gel electrophoresis and used as competitive inhibitors in the ELISA. Autoradiography of the gel showed an intense band corresponding to the 4',5'-monoadduct and two weaker unidentified bands. Antibody 7E3 reacted only with the 4',5'-monoadduct band as would be expected since this photoadduct was the principal photoadduct in the original antigen.
Miolo
G
G
Department of Pharmaceutical Sciences, Padua University, Italy.
Stefanidis
M
M
Santella
R M
RM
Dall'Acqua
F
F
Gasparro
F P
FP
D000911:Antibodies, Monoclonal / Q000032:analysis / Q000096:biosynthesis*; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000187:drug effects*; D004249:DNA Damage; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D011564:Furocoumarins / Q000378:metabolism*; D006868:Hydrolysis; D010777:Photochemistry; D013056:Spectrophotometry, Ultraviolet
D000911:Antibodies, Monoclonal; D011564:Furocoumarins; D004247:DNA; C041621:4,4',6-trimethylangelicin