Deoxyribonucleic acid replication in single cells and chromosomes by immunologic techniques

The identification of cells in a population which is undergoing DNA replication has been accomplished in the past by autoradiographic techniques utilizing ³H thymidine. The autoradiographic methods, however, lack cytologic resolution and require relatively long periods of exposure to produce sufficient silver grains for detection. In addition, for the purposes of automated flow analysis, autoradiographic techniques are not applicable. Antibodies to 5 bromodeoxyuridine (BrdU) or iododeoxyuridine may be used to identify cells or regions of chromosomes in which de novo deoxyribonucleic acid synthesis has occurred. The antibodies to BrdU were produced in rabbits by injection of the antigen, a conjugate between bovine serum albumin and bromouridine (BrU), or iodouridine. Specific antibodies were produced by affinity chromatography on AH Sepharose 4B to which had been coupled BrU. Anti BrU cross reacts with iododeoxyuridine. Indirect antibody techniques have been used to monitor deoxyribonucleic acid synthesis in nuclei; anti BrdU treatment was followed by goat anti rabbit immunoglobulin G labeled with either fluorescein or horseradish peroxidase. By use of these techniques, labeling indices were determined in cell cultures which had been pulsed with ³H BrdU. The immunologic technique compared favorably with the autoradiographic methods performed concurrently on the same cultures. Metaphase chromosomes from synchronous CHO cells which had been pulse labeled with BrdU at different time intervals during S phase were subjected to these immunologic procedures. Chromosome banding was observed with both the fluorescence and peroxidase methods. Chromosomes from cells not containing BrdU did not exhibit banding.