Polyclonal antibodies elicited against trans-diamminedichloroplatinum(II)-modified DNA and their application in nonradioactive hybridization experiments.
Polyclonal antibodies that bind selectively to adducts formed with DNA by chemotherapeutically inactive trans-diamminedichloroplatinum (II) [trans-[Pt(NH3)2Cl2]) were produced by immunization with calf-thymus double-helical DNA modified by trans-[Pt(NH3)2Cl2] at a ratio of bound platinum/nucleotide (rb) of 0.1. High selectivity was obtained by separation of the antibodies from the antiserum with the aid of affinity chromatography on a Sepharose column. The antibodies were competitively inhibited in an ELISA assay by 25 pM trans-[Pt(NH3)2Cl2] bound to double-helical DNA and 2.5 pM trans-[Pt(NH3)2Cl2] bound to denatured DNA (rb = 0.1). The conversion of monofunctional adducts, formed on DNA at the early stage of its interaction with trans-[Pt(NH3)2Cl2], to bifunctional lesions, decreased the ability of the modified DNA to competitively inhibit these antibodies. They did not cross-react with unmodified, denatured DNA, but they reacted with diethylenetriamine-chloroplatinum(II)-chloride-modified double-helical DNA and with double-helical DNA treated with cis-diamminedichloroplatinum(II) for a short time (10 min). The results of this work best fit a model in which one of the major antigenic determinants of double-helical DNA modified by trans-[Pt(NH3)2Cl2] is the platinum atom coordinated in a monodentate or bidentate manner with non-paired nucleotide residues or perhaps a short segment of single-stranded DNA which occurs around the platination site. Nucleic acids modified by trans-[Pt(NH3)2Cl2] can be used as immuno-probes in hybridization experiments.
Brabec
V
V
Institute of Biophysics, Czechoslovak Academy of Sciences, Brno.
Vrana
O
O
Kiseleva
V I
VI
Poverenny
A M
AM
D000818:Animals; D000917:Antibody Formation; D001667:Binding, Competitive; D002945:Cisplatin / Q000276:immunology* / Q000378:metabolism; D004247:DNA / Q000276:immunology* / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D009691:Nucleic Acid Denaturation; D009693:Nucleic Acid Hybridization; D011048:Polarography / Q000379:methods; D011817:Rabbits
C034697:transplatin; D004247:DNA; D002945:Cisplatin