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Antibodies against cisplatin-modified DNA and cisplatin-modified (di)nucleotides
P M Terheggen1, B G Floot, E L Lempers
1Division of Chemical Carcinogenesis, Netherlands Cancer Institute (Antoni van Leeuwenhoek Huis), Amsterdam.
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.
Insights
Researchers developed specific antibodies to detect cis-diamminedichloroplatinum-(II) (cis-DDP) DNA adducts. Antiserum NKI-A59 enables in situ detection of cis-DDP-DNA adducts at clinically relevant platinum levels.
Area of Science:
- Biochemistry
- Molecular Biology
- Immunology
Background:
- Cytotoxic effects of cis-diamminedichloroplatinum-(II) (cis-DDP) are linked to DNA binding.
- Accurate detection of cis-DDP-DNA adducts is crucial for understanding its mechanism of action.
- Existing methods for studying cis-DDP-DNA binding rely on specific antibodies.
Purpose of the Study:
- To develop and characterize novel antisera for detecting cis-DDP-modified DNA.
- To evaluate the utility of these antisera in enzyme-linked immunosorbent assays (ELISA) and immunocytochemical assays.
- To identify antibodies suitable for detecting cis-DDP-DNA adducts at clinically relevant levels.
Main Methods:
- Raised four rabbit antisera against cis-DDP-modified DNA and (di)nucleotides.
- Assessed antibody reactivity using ELISA and quantitative immunocytochemical assays.
- Determined affinity for various platinum compounds, DNA modification levels, and DNA types.
Main Results:
- Antiserum NKI-A59 demonstrated high affinity for cis-DDP- and CBDCA-modified DNA, enabling detection at low fmol levels.
- NKI-A59 showed specificity for cis-DDP-modified DNA, with binding decreasing at lower modification levels.
- Antisera NKI-A68, NKI-A10, and NKI-A39 were effective for analyzing digested cis-DDP-modified DNA in ELISA.
Conclusions:
- NKI-A59 is the first described antiserum suitable for in situ detection of cis-DDP-DNA adducts at clinically relevant platinum levels.
- NKI-A68, NKI-A10, and NKI-A39 are valuable for platinum-DNA adduct analysis of digested DNA.
- NKI-A59 facilitates platinum-DNA adduct detection at the single-cell level via immunocytochemistry.