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

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 c...

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Bibliographic Details
Published in:PloS one 2015-07, Vol.10 (7), p.e0132393-e0132393
Main Authors: Ligasová, Anna, Liboska, Radek, Rosenberg, Ivan, Koberna, Karel
Format: Article
Language:English
Subjects:
Affinity
Analysis
Antibodies
Antibodies, Monoclonal - metabolism
Biochemistry
Bromodeoxyuridine
Bromodeoxyuridine - metabolism
Cell cycle
Clone Cells
Cloning
Copper
Cytotoxicity
Degradation
Deoxyribonucleic acid
DNA
DNA - metabolism
DNA polymerase
DNA probes
HCT116 Cells
HeLa Cells
Humans
Idoxuridine - analogs & derivatives
Idoxuridine - metabolism
Immunoglobulins
Laboratories
Medicine
Monoclonal antibodies
Oligonucleotides
Organic chemistry
Peptide Mapping
Reactivity
Studies
Toxicity
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Summary: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.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0132393