Improved high-affinity monoclonal antibody to iododeoxyuridine

Area of Science:

• Biochemistry
• Molecular Biology
• Immunology

Background:

• Monoclonal antibodies (Mabs) are crucial tools for detecting modified DNA bases.
• Bromodeoxyuridine (BrdUrd) and iododeoxyuridine (IdUrd) are thymidine analogs used in various biological studies.
• Existing Mabs often require high substitution frequencies for reliable DNA detection.

Purpose of the Study:

• To evaluate three Mabs (IU-1, IU-4, B-44) for their ability to recognize BrdUrd- and IdUrd-substituted DNA, nucleotides, and nucleosides.
• To characterize the binding affinity and specificity of the novel Mab IU-4.
• To determine the suitability of IU-4 as a reagent for quantitative labeling of halogenated thymidine analogs.

Main Methods:

• Enzyme immunoassays (ELISA) were employed to assess antibody binding.
• Flow cytometry was used for cellular analysis of antibody recognition.
• Competition assays were performed to determine antibody specificity and affinity.

Main Results:

• The novel Mab IU-4 demonstrated a strong preference for IdUrd over BrdUrd.
• IU-4 recognized IdUrd in DNA at substitution frequencies as low as 1 in 1,000 bases.
• Unlike IU-1 and B-44, IU-4's binding was independent of IdUrd residue spacing.

Conclusions:

• Mab IU-4 exhibits high affinity and specificity for IdUrd-substituted DNA.
• IU-4 is a superior reagent for the quantitative labeling of halogenated thymidine analogs in whole cells.
• The findings facilitate more sensitive detection of DNA modifications using specific Mabs.