Three monoclonal antibodies (Mabs), IU-1, IU-4, and B-44 were evaluated in enzyme immunoassays (ELISA) and by flow cytometry for their abilities to recognize bromodeoxyuridine (BrdUrd)-and iododeoxyuridine (1dUrd)-substituted DNA's, nucleotides, and nucleosides. IU-4 is a new Mab, derived from mice immunized with 5-iodo-2'-deoxyuridine-5'-monophosphate (IdUMP) conjugated through the phosphate group to albumin. This immunogen was selected to resemble IdUMP in DNA. In competition ELISA assays, IU-4 prefers IdUrd to BrdUrd and prefers halogenated nucleotides over the corresponding nucleosides. In both ELISA and flow analysis, IU-4 recognizes IdUrd in DNA at substitution frequencies at least as low as one IdUrd one per 1,000 normal bases. The high affinity of IU-4 for IdUrd-DNA contrasts with IU-1 and B-44, which show a strong binding dependency on the frequency of base substitution and require DNA that is essentially fully substituted with BrdUrd for binding in bath flow and ELISA assays. The high affinity of IU-4 for IdUrd in DNA and its independence of IdUrd residue spacing make it a superior reagent for the quantitative labeling of halogenated thymidine analogues in whole cells.Key terms: Fj-Rromo-Z'-deoxyuridine, flow cytometry, 5-iodo-2'-deoxyuridine, ELISA, DNA immunoassay, cell kineticsImmunoassays for DNA substituted with the halogenated nucleosides bromodeoxyuridine (BrdUrd) and iododeoxyuridine (IdUrd) are widely used in applications such as the quantification of cell cycle kinetics, the detection of unscheduled DNA synthesis, and the isolation of nascent DNA (7,15). Several monoclonal antibodies (Mabs) suitable for these assays have been reported, and all share some common developmental histories and properties (13,14,25,28,29). In all cases, the immunogen was a halogenated ribouracil conjugated to a carrier protein using the periodatehorohydride method developed by Erlanger and Beiser (11). This conjugation method changes the ribose to a six-membered ring containing nitrogen. The epitope in common with DNA is limited to the halogenated uracil. The choice of bromine or iodine in the base appears not to influence antibody specificity, since, where reported, the resulting antibodies react equally with both IdUrd and BrdUrd. The antibodies are of generally low affinity (Ka's of approximately lo6 M-for BrdUrd in solution) and only recognize the altered base in single-stranded DNA (ss-DNA), not in native DNA. For many applications, these characteristics have not been limiting, and the antibodies have found wide use.For a number of experiments, however, the antibodies are unsuitable. For example, we report here that one disadvantge of Mabs IU-1(30) and B-44 (14) is that both require a high local linear density of BrdUrd substitution in the DNA strand in order to bind. The magnitude of binding depends both on the total amount of incorporated BrdUrd as well as the spacing between BrdUrd residues. High linear density is readily obtained for assays on cell lines grown in vitro but is harder to achieve when tissues...
