Improved high‐affinity monoclonal antibody to lododeoxyuridine

Vanderlaan, Martin; Watkins, Bruce E.; Thomas, C.; Dolbeare, Frank; Stanker, Larry H.

doi:10.1002/cyto.990070602

Cited by 60 publications

(20 citation statements)

References 25 publications

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“…These factors include the degree of DNA denaturation, the concentration and affinity of monoclonal antibodies, the fluorescent probe used, and the number of fluorescent molecules attached to the secondary antibody. Vanderlaan et al have documented the high affinity of IU-4 with reliable detection of BrdUrd at densities 5 2.5% (11). Loweraffinity monoclonal antibodies might be associated with a lesser degree of BrdUrd concentration independence.…”

Section: Discussionmentioning

confidence: 99%

Bromodeoxyuridine incorporation into DNA of human leukemia cells is not concentration dependent

et al. 1990

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Leukemia blasts isolated from bone marrow aspirates of 44 adults with acute leukemia were incubated for 1 h with 0.008-32 pM bromodeoxyuridine (BrdUrd). After dual labeling with monoclonal anti-BrdUrd antibodies and propidium iodide, the cells were analyzed by flow cytometry. Percent labeled cells and intensity of labeling were similar over concentrations of BrdUrd ranging from 0.8-32 pM-a 40-fold range. Therefore, despite potential interpatient variability in nucleoside pharmacokinetics, commonly used doses of BrdUrd which are intended to achieve steady-state plasma concentrations in the 8.0 pM range can be expected to provide a reliable estimate of the S-phase fraction. Key terms: Cell kinetics, acute leukemia, concentration independence, flow cytometryMeasurements of incorporation of bromodeoxyuridine (BrdUrd) into DNA have provided a useful assessment of DNA synthesis in leukemia cells in vitro and in vivo (3)(4)(5)8,9,12). The amount of BrdUrd incorporated into DNA can be quantified by either flow cytometric (3-5,12) or microscopic techniques (8,9). If BrdUrd incorporation is to be a reliable in vivo measure, possible concentration-dependent artifacts must be avoided. Although interpatient variations in BrdUrd pharmacokinetics have not been extensively evaluated, continuous infusions of a fixed dose of the nucleoside cytosine arabinoside in leukemia patients can result in a 10-fold variation in plasma concentration a t steady state (1). It is expected that similar variations in plasma concentrations of other nucleosides such as BrdUrd might occur. BrdUrd doses (200 mgim' over 1 h) used in in vivo cytokinetic studies yield concentrations in the 8 pM range (5). A recent trial employed a lower dose of 100 mgim' (8); although pharmacokinetics were not evaluated in this study, elimination is expected to be linear (10). The toxicities of BrdUrd are dose dependent, but are not expected with the doses administered for cytokinetic studies (6).In an attempt to establish a range of concentrations over which BrdUrd incorporation into DNA of human leukemia cells is constant, the following experiment was performed. MATERIALS AND METHODSIsolation of Leukemia Cells A separate marrow aspirate from the posterior iliac crest of adult patients with acute leukemia was collected in heparinized syringes and resuspended in 30 cc of ice-cold RPMI medium 1640 (Gibco, Grand Island, NY). This suspension was layered over 15 ml of FicollPaque (Pharmacia, Picataway, NJ; density = 1.077) and centrifuged at 1,OOOg for 20 min. Cells at the plasma-Ficoll interface were harvested and washed twice with 50 ml of RPMI and then resuspended in 2 ml of RPMI with 10% fetal calf serum (FCS)

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Section: Discussionmentioning

confidence: 99%

Bromodeoxyuridine incorporation into DNA of human leukemia cells is not concentration dependent

et al. 1990

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“…Recently, monoclonal antibodies, Br-3 that recognizes BrdU specifically [4] and IU-4 that recognizes both BrdU and iododeoxyuridine (IUdR) [31], have been developed.…”

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Kinetic Analysis of Tumor Cells in Bone and Soft Tissue Sarcomas.

Morimoto

Matsumoto

Chano

et al. 1997

Acta Histochem. Cytochem

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“…They found that the monoclonal antibody B44 (5) was minimally reactive to CldUrd after incubation in a highsalt buffer. To stain CldUrd specifically, they used an antibody with minimal reactivity to IdUrd, similar to the Br3 antibody described previously by Vanderlaan et al (8). Their procedure involved indirect staining of both B44 and the anti-CldUrd-specific antibody.…”

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confidence: 99%

Flow cytometric analysis of two incorporated halogenated thymidine analogues and DNA in a mouse mammary tumor grown in vivo

et al. 1993

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A technique was developed for the staining of nuclei for DNA using propidium iodide, and incorporated chlorodeoxyuridine (CldUrd) and iododeoxyuridine (IdUrd) using two monoclonal antibodies that showed negligible crossreactivity. The mouse mammary solid tumor MCaK was labeled in vivo by intraperitoneal injection of the nucleosides. Tumor cell nuclei were stained after isolation from ethanol-fixed solid tumor tissue and acid denaturation. The Br3 antibody, which specifically recognizes CldUrd, was applied first, followed by indirect staining with goat anti-mouse phycoerythrin. The direct fluorescein isothiocyanate conjugate of the B44 antibody, which specifically recognizes IdUrd, was then applied. In the direct conjugate form this antibody reacted only minimally with CldUrd. The nuclei were then stained with propidium iodide. With this dye combination the coefficients of variations of the DNA histograms were consistently in the 2-4% range. Two other dye combinations were compared. The propidium iodide/phycoerythrin/fluorescein isothiocyanate dye combination was the simplest because of the compatibility with single laser flow cytometry. Key terms: Propidium iodide, chlorodeoxyuridine, iododeoxyuridine There are several potential applications for the simultaneous and specific quantification of two different halogenated nucleoside analogues in double-labeled DNA. These include direct measurement of recruitment, simultaneous determination of growth fraction and cell kinetics, and more accurate measurement of potential doubling time (T&.A number of bromodeoxyuridine (BrdUrd)-reactive monoclonal antibodies are now commercially available. These antibodies bind differently to the other halogenated thymidine analogues, chlorodeoxyuridine (CldUrd) and iododeoxyuridine (IdUrd). Recently, Bakker et al. (2) described a n indirect method for staining nuclei double-labeled with CldUrd and IdUrd. They found that the monoclonal antibody B44 (5) was minimally reactive to CldUrd after incubation in a highsalt buffer. To stain CldUrd specifically, they used an antibody with minimal reactivity to IdUrd, similar to the Br3 antibody described previously by Vanderlaan et al. (8). Their procedure involved indirect staining of both B44 and the anti-CldUrd-specific antibody. The indirect staining, combined with mandatory incubation and wash in a high-salt buffer, makes the procedure relatively long. Moreover, while Bakker et al. (2) demonstrated simultaneous staining of CldUrd and IdUrd in DNA, they did not show any results of three color flow cytometric analysis.We describe here our investigation of different procedures for three-color analysis of two incorporated halogenated thymidine analogues and DNA. We compared three staining techniques, shortened the staining procedure considerably, and adapted the method for solid tumors labeled in vivo. The simplest method involved the dye combination of phycoerythrin (PE)-tagged second antibody to Br3, fluorescein isothiocyanate (F1TC)-tagged B44, and propidium iodide (PI).

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Improved high‐affinity monoclonal antibody to lododeoxyuridine

Cited by 60 publications

References 25 publications

Bromodeoxyuridine incorporation into DNA of human leukemia cells is not concentration dependent

Bromodeoxyuridine incorporation into DNA of human leukemia cells is not concentration dependent

Kinetic Analysis of Tumor Cells in Bone and Soft Tissue Sarcomas.

Flow cytometric analysis of two incorporated halogenated thymidine analogues and DNA in a mouse mammary tumor grown in vivo

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