title: Antibodies against cisplatin-modified DNA and cisplatin-modified (di)nucleotides.
abstract: Cytotoxic effects of cis-diamminedichloroplatinum-(II) (cis-DDP) are thought to be mediated by binding to DNA. Studies on binding of cis-DDP to cellular DNA rely heavily on the availability of specific antibodies. We therefore raised and characterized four rabbit antisera: one against cis-DDP-modified DNA (antiserum NKI-A59) and three others against the cis-DDP-modified (di)nucleotides cis-Pt(NH3)2d(pApG) (NKI-A68), cis-Pt(NH3)2d(GMP)2 (NKI-A10), and Pt(NH3)3dGMP (NKI-A39). Reactivities to platinum compounds were determined in an enzyme-linked immunosorbent assay (ELISA) and in a quantitative immunocytochemical assay. In the ELISA, NKI-A59 showed a high affinity for DNA heavily substituted with either cis-DDP or CBDCA [cis-diammine(1,1-cyclobutanedicarboxylato)platinum(II)]; amounts of platinum per well giving 50% inhibition (IA50) were as low as 15 and 76 fmol, respectively. NKI-A59 also showed affinity to cis-DDP-modified poly[d(G-C)].poly[d(G-C)], poly(dC), and poly(dG). No affinity was found for trans-DDP [trans-diamminedichloro-platinum(II)]-modified DNA, enzymatically digested cis-DDP-DNA, or cis-DDP-DNA, or cis-DDP-modified poly(dA).poly(dT), oligo(dA)15.oligo(dT)15, oligo(dG)21, oligo(dG)42, or oligo(dAAAG)10. The efficiency of binding to cis-DDP-DNA decreased with decreasing DNA modification levels. Although other cis-DDP-DNA- and cis-DDP-(di)nucleotide-specific antisera have been identified, NKI-A59 is the first antiserum described that is suitable for the in situ detection of cis-DDP-DNA adducts at clinically relevant platinum levels. Adduct-specific immunostaining signals in cultured RIF-1 cells or rat liver paralleled platinum-DNA binding as measured by atomic absorption spectroscopy. The antisera NKI-A68, NKI-A10, and NKI-A39 showed high affinity for their corresponding haptens and varying affinity for non-hapten cis-DDP-DNA adducts. Their affinity for digested cis-DDP-modified DNA was up to 30 times that for intact cis-DDP-DNA. Neither NKI-A68 nor NKI-A10 resulted in specific immunocytochemical staining of cis-DDP-DNA adducts. We conclude that NKI-A68, NKI-A10, and NKI-A39 are suitable for platinum-DNA adduct analysis of digested DNA in ELISA and that NKI-A59 is suitable for platinum-DNA adduct detection at the single-cell level using immunocytochemical methods.
authors.affiliation: Division of Chemical Carcinogenesis, Netherlands Cancer Institute (Antoni van Leeuwenhoek Huis), Amsterdam.
authors.forename: P M
authors.initials: PM
authors.lastname: Terheggen
authors.forename: B G
authors.initials: BG
authors.lastname: Floot
authors.forename: E L
authors.initials: EL
authors.lastname: Lempers
authors.forename: O
authors.initials: O
authors.lastname: van Tellingen
authors.forename: A C
authors.initials: AC
authors.lastname: Begg
authors.forename: L
authors.initials: L
authors.lastname: den Engelse
mesh_terms: D000818:Animals; D000906:Antibodies / Q000032:analysis / Q000302:isolation & purification*; D000918:Antibody Specificity / Q000187:drug effects / Q000276:immunology; D002478:Cells, Cultured / Q000187:drug effects / Q000276:immunology; D002945:Cisplatin / Q000494:pharmacology*; D004247:DNA / Q000032:analysis / Q000187:drug effects* / Q000276:immunology* / Q000302:isolation & purification; D004797:Enzyme-Linked Immunosorbent Assay; D007106:Immune Sera / Q000032:analysis / Q000302:isolation & purification; D007150:Immunohistochemistry; D008297:Male; D051379:Mice; D009841:Oligonucleotides / Q000032:analysis / Q000276:immunology*; D011119:Polynucleotides / Q000032:analysis / Q000276:immunology*; D011817:Rabbits; D051381:Rats; D011919:Rats, Inbred Strains
chemical_list: D000906:Antibodies; D007106:Immune Sera; D009841:Oligonucleotides; D011119:Polynucleotides; D004247:DNA; D002945:Cisplatin