title: The Fingerprint of Anti-Bromodeoxyuridine Antibodies and Its Use for the Assessment of Their Affinity to 5-Bromo-2'-Deoxyuridine in Cellular DNA under Various Conditions.
abstract: We have developed a simple system for the analysis of the affinity of anti-bromodeoxyuridine antibodies. The system is based on the anchored oligonucleotides containing 5-bromo-2'-deoxyuridine (BrdU) at three different positions. It allows a reliable estimation of the reactivity of particular clones of monoclonal anti-bromodeoxyuridine antibodies with BrdU in fixed and permeabilized cells. Using oligonucleotide probes and four different protocols for the detection of BrdU incorporated in cellular DNA, we identified two antibody clones that evinced sufficient reactivity to BrdU in all the tested protocols. One of these clones exhibited higher reactivity to 5-iodo-2'-deoxyuridine (IdU) than to BrdU. It allowed us to increase the sensitivity of the used protocols without a negative effect on the cell physiology as the cytotoxicity of IdU was comparable with BrdU and negligible when compared to 5-ethynyl-2'-deoxyuridine. The combination of IdU and the improved protocol for oxidative degradation of DNA provided a sensitive and reliable approach for the situations when the low degradation of DNA and high BrdU signal is a priority.
authors.affiliation: Institute of Molecular and Translational Medicine, Faculty of Medicine, Palacký University, Olomouc, Czech Republic.
authors.forename: Anna
authors.initials: A
authors.lastname: Ligasová
authors.affiliation: Oligonucleotide group, Institute of Organic Chemistry and Biochemistry CAS, v.v.i., Prague, Czech Republic.
authors.forename: Radek
authors.initials: R
authors.lastname: Liboska
authors.affiliation: Oligonucleotide group, Institute of Organic Chemistry and Biochemistry CAS, v.v.i., Prague, Czech Republic.
authors.forename: Ivan
authors.initials: I
authors.lastname: Rosenberg
authors.affiliation: Institute of Molecular and Translational Medicine, Faculty of Medicine, Palacký University, Olomouc, Czech Republic.
authors.forename: Karel
authors.initials: K
authors.lastname: Koberna
mesh_terms: D000911:Antibodies, Monoclonal / Q000378:metabolism*; D001973:Bromodeoxyuridine / Q000378:metabolism*; D002999:Clone Cells; D004247:DNA / Q000378:metabolism*; D045325:HCT116 Cells; D006367:HeLa Cells; D006801:Humans; D007065:Idoxuridine / Q000031:analogs & derivatives / Q000378:metabolism; D010449:Peptide Mapping
chemical_list: D000911:Antibodies, Monoclonal; C029954:5'-deoxy-5'-iodouridine; D004247:DNA; D001973:Bromodeoxyuridine; D007065:Idoxuridine