Interaction of monoclonal antibodies directed against bromodeoxyuridine with pyrimidine bases, nucleosides, and DNA

Although antibodies directed against bromodeoxyuridine (BrdU) are being used in both clinical and basic research laboratories as tools to study and monitor DNA synthesis, little is known about the epitopes with which they react. Four monoclonal antibodies directed against BrdU were produced and were characterized to learn more about the epitopes on BrdU which are important for antibody recognition, to identify compounds other than BrdU which react with the antibodies and which might interfere with immunologic assays for BrdU, and to characterize the reaction of these antibodies with BrdU-containing DNA. By radioimmunoassays, the antibodies generally reacted well with 5-iododeoxyuridine, 5-fluorodeoxyuridine, and 5-nitrouracil. However, none of the antibodies reacted well with uridine--indicating that a substituent on uridine C5 was essential for antibody reactivity--or with 5-bromo- or iodo-cytosine, indicating that the region around pyrimidine C4 is important for antibody recognition. Although the antibodies reacted with 5-halogen-substituted uracil bases, the antibodies reacted much better with the corresponding halogenated nucleosides, indicating that the sugar moiety was important for recognition. The presence of a triphosphate group on C'5 of BrdU (i.e., BrdUTP) did not detectably alter antibody recognition. Three of the antibodies reacted only with purified DNA containing BrdU, whereas one antibody, which exhibited a weak interaction with thymidine, also reacted with BrdU-free DNA. S1 nuclease treatment of purified DNA suggested that all four monoclonal antibodies reacted exclusively with single-stranded regions of BrdU-containing DNA. Comparison of detecting DNA synthesis by [3H]TdR incorporation followed by autoradiography with that by BrdU incorporation followed by indirect immunofluorescence indicated that the latter technique was both an accurate and a sensitive measure of DNA synthesis.     

Immunochemical detection of 7-methylguanine residues in nucleic acids

The ability of specific antibodies to react with 7-methylguanine residues in nucleic acids was investigated. Anti-7-methylguanine specific antibodies precipitated polymers of poly-guanylic acid which were methylated to an extent of 35 or 70% at the N-7 position of guanine, indicating that these antibodies could readily detect 7-methylguanine residues in a polynucleotide. This reaction was proportional to the total amount of 7-methylguanine present, suggesting further that quantitation of these residues is possible. To determine the minimal amount required for detection, varying amounts of 7-methylguanine were introduced into calf thymus DNA by alkylation with dimethyl sulfate. While showing no reaction with denatured nonalkylated DNA, the reaction of antibodies with alkylated DNA was proportional to the amount of 7-methylguanine in the preparations. Moreover, the antibodies appeared to detect differences in the distribution of 7-methylguanine residues in extensively methylated DNA. Precipitation was observed with DNA containing as little as one 7-methylguanine residue per 300 nucleotides, suggesting that these antibodies can be used to detect biologically significant levels of 7-methylguanine in viral and cellular nucleic acids.     

Visualization of Z sequences in form V of pBR322 by immuno-electron microscopy.

Form V DNA has been prepared from pBR322 DNA by annealing covalently closed complementary single strands. Specific rabbit antibodies to Z-DNA were shown by radioimmunoassay and electron microscopy to react with form V DNA of pBR322. The bound antibodies were visualized either directly (on synthetic polynucleotides in Z-form), or after reaction with goat anti-rabbit immunoglobulin labeled with ferritin (on form V DNA).     

The reactivity of lupus erythematosus serum with native and denatured DNA of various origins]

The serum of a female suffering from Lupus erythematosus visceralis was investigated by complement fixation for the reaction with native and denatured DNA's of various base composition. The reaction with native DNA is independent on the (G+C)-content of the DNA. It is apparent that the responsible antibodies react with determinants of the helical conformation, which are identical in the various DNA-molecules. Quantitative differences are found with denatured DNA's. The strongest complement fixation is observed with (G+C)-rich denatured DNA. The reaction with denatured DNA is only partially inhibited by DNA digest. These antibodies obviously react with sequential determinants containing bases. Therefore, they are induced by a different mechanism of sensitization.     

Murine monoclonal anti-DNA autoantibodies bind to endogenous bacteria

Several bacterial species (including Streptococcus faecalis, Bacillus cereus, Staphylococcus aureus, and Escherichia coli) were tested for their ability to react with monoclonal anti-DNA antibodies that were derived from MRL-lpr/lpr mice. S. faecalis reacted with 8/15 of such antibodies. The binding was unaffected by DNase, but it was competitively inhibited by DNA. F(ab')2 fragments of the monoclonal antibodies reacted with the bacteria, but Fc fragments did not. Phospholipids extracted from the bacterial cells were able to bind to three representative anti-DNA antibodies that also bound to whole bacteria. The results suggest that bacterial phospholipids might provide an immunogenic stimulus for the production of antibodies that cross-react with DNA. We propose that some anti-DNA auto-antibodies and anti-bacterial antibodies evolve from a restricted group of antibodies with high avidity for the phosphodiester groups that occur in DNA and bacterial cells walls.     

Monoclonal antibodies to the rat liver glucocorticoid receptor.

Monoclonal antibodies against the 90 000 mol. wt. form of the activated rat liver glucocorticoid receptor were generated from mice immunized with a partially purified receptor preparation. The screening assay was based on the precipitation of liver cytosol, labelled with [3H]triamcinolone acetonide, with monoclonal antibodies bound to immobilized rabbit anti-mouse IgG. Out of 102 hybridomas obtained, 76 produced immunoglobulin and eight of them were found to react with the receptor molecule. Only one of the positive clones secreted IgG whereas the other seven produced IgM. The complexes of receptor and antibodies were identified by sucrose density gradient centrifugation. All seven monoclonal antibodies tested reacted with the 90 000 mol. wt. form of the receptor but not with the 40 000 mol. wt. form that contains the steroid and DNA binding domains. None of the monoclonal antibodies interfered with the binding of the receptor to DNA cellulose, thus suggesting that the antigenic determinants are located in a region of the receptor that is not directly implicated in either steroid binding or DNA binding. These antigenic determinants were common to glucocorticoid receptors from several tissues of the rat, whereas glucocorticoid receptors from other species react only with some of the antibodies.     

Antibodies to DNA

Antibodies that recognize specific conformational variations of DNA structure provide sensitive reagents for testing the extent to which such conformational heterogeneity occurs in nature. A most dramatic recent example has been the development and application of antibodies to left-handed Z-DNA. They provided the first identification of Z-DNA in fixed nuclei and chromosomes, and of DNA sequences that form Z-DNA under the influence of supercoiling. Antibodies have also been induced by chemically modified DNA and by synthetic polydeoxyribonucleotides that differ from the average B-DNA structure. These antibodies recognize only the features that differ from native DNA. In most experiments, native DNA itself is not immunogenic. Antibodies that do react with native DNA occur in sera of patients with autoimmune disease, but even monoclonal anti-DNA autoantibodies usually react with other polynucleotides as well. Anti-DNA antibodies, especially those of monoclonal origin, provide a model for the study of protein-nucleic acid recognition.     

"Masked" autoantibodies from the serum of healthy blood donors cross-reacting with DNA and bacterial lipopolysaccharides

The "masked" autoantibodies with polyspecific activity were found in the serum of healthy donors. It is shown that these antibodies are able to react with antigens after the ion exchange chromatography with QAE-sephadex. It was found that these "masked" antibodies are able to cross-react with DNA and lipopolysaccharides of wide-spread species of bacteria. The possible role of these antibodies as in protection of organism from the external antigens as in the origin of autoimmune diseases is discussed.     

Monoclonal autoantibodies to subnucleosomes from a MRL/Mp(-)+/+ mouse. Oligoclonality of the antibody response and recognition of a determinant composed of histones H2A, H2B, and DNA.

MRL/Mp(-)+/+ mice produce antinuclear antibodies and develop a spontaneous autoimmune syndrome with lupus-like nephritis. We obtained a panel of seven histone-reactive IgG mAb from a single MRL/Mp(-)+/+ mouse. These antibodies do not react significantly with DNA or individual histones, but bind strongly to the histone H2A-H2B dimer and even more strongly to the H2A-H2B-DNA complex. These antibodies also bind to whole nuclei when tested by immunofluorescence, indicating that they recognize an epitope accessible in chromatin. The V region sequences of these antibodies have been determined. The H chain third complementarity-determining regions of these antibodies are similar to those found in anti-DNA antibodies even though the antibodies in our panel do not react with DNA in the absence of histones, suggesting that DNA is part of the subnucleosome epitope. Several of these antibodies are clonally related, supporting the hypothesis that the activation of these clones is Ag-driven. Analysis of the sequences of these antibodies indicates that they derive from autoreactive B cells that were clonally expanded and whose V region genes have undergone numerous somatic mutations.     

Characterization of monoclonal antibodies to bromodeoxyuridine

The characteristics of three mouse monoclonal antibodies to halogenated uridine derivatives are presented. Two, IU-1 and IU-2, are produced by hybridomas derived in our laboratory, and the third is the B-44 hybridoma described by Gratzner (7) and obtained commercially from Becton-Dickinson Monoclonal Center. Hybridomas IU-1 and IU-2 were derived from the fusion of spleen cells from a Biozzi High Responder mouse immunized with iododeoxyuridine (IdUrd) conjugated to bovine serum albumin and SP2/0 mouse myeloma cells. This paper presents methods and results for enzyme-linked immunosorbent assays (ELISA) against whole cells labeled with bromodeoxyuridine (BrdUrd), ELISA against BrdUrd-labeled DNA, and a competition ELISA for free BrdUrd. All three antibodies show similar binding affinities and specificities. The IU antibodies react with BrdUrd and IdUrd when the nucleosides are either free in solution or incorporated into single-stranded DNA (ss-DNA). The antibodies do not recognize either halogenated base in double-stranded DNA (ds-DNA), nor do they react with uracil or bromocytidine. Weak binding to thymidine, 5-fluorodeoxyuridine, and unsubstituted ss-DNA occurs.     

Different reactivity of Z-DNA antibodies with human chromosomes modified by actinomycin D and 5-bromodeoxyuridine

Summary Antibodies against Z-DNA react with fixed metaphase chromosomes of man and other mammals. Indirect immunofluorescence staining shows that chromosomal segments corresponding to R- and T-bands preferentially fix Z-DNA antibodies. In this work Z-DNA antibodies were used as a probe for DNA conformation in euchromatin of fixed human chromosomes whose condensation or staining were modified by actinomycin D (AMD) and by 5-bromodeoxyuridine (BrdU). Treatments with AMD and BrdU were performed to induce a G-banding by modification of chromosomal segments corresponding to R- and T-bands. Long BrdU treatments were used to induce asymmetrical and partially undercondensed chromosomes by substitution of thymidine in one or both DNA strand. Our results show a clear difference of Z-DNA antibodies reactivity after AMD or BrdU treatment. The G-banding obtained after AMD treatment is not reversed by Z-DNA antibodies staining since these antibodies bind very weakly to the undercondensed R-bands. On the other hand, the G-banding obtained by BrdU is completely reversed giving typical R-banding, as on untreated chromosomes. For asymmetrical chromosomes an R-, T-banding pattern is always observed but there is a decrease of the fluorescence intensity proportional to the degree of BrdU incorporation. We conclude that AMD treatment greatly disturbs Z-DNA antibodies binding suggesting a change in DNA conformation, whereas BrdU treatments do not suppress but only weaken the specific binding of Z-DNA antibodies on R- and T-bands. The direct involvement of thymidine substitution in DNA sequences recognized by Z-DNA antibodies is discussed.     

Characterization of anti-ApApA antibodies

Antibodies to a trinucleotide, ApApA, were prepared by injecting bovine serum albumin conjugated with ApApA into rabbits. The specificities of the antibodies were determined by estimating the inhibiton of the binding of [14C]ApApA to the antibodies by various nonradioactive mono-, oligo-, and polynucleotides, using the ammonium sulfate precipitation method. The antibodies were found to react with ApApN sequences and oligoadenylic acids, but also reacted slightly with polyadenylic acid, RNA and DNA. Significant crossreactions were observed with other oligonucleotides containing adenosine.     

Monoclonal antibodies neutralizing mammalian DNA topoisomerase I activity

We have isolated three different monoclonal antibodies specific for mammalian type-I DNA topoisomerase. The antibodies react with three closely adjacent epitopes located in a central section of the enzyme (between amino acid residues 344 and 483). Two of the antibodies inhibit an early step of the nicking/closing pathway. We provide evidence showing that the antibodies do not block the association of the enzyme with DNA. The antibodies are useful for immunocytochemical investigation and for further exploration of the biochemical function of mammalian type-I DNA topoisomerase.     

Monoclonal antibodies that recognize lacto-N-fucopentaose III (CD15) react with the adhesion-promoting glycoprotein family (LFA-1/HMac-1/gp 150,95) and CR1 on human neutrophils

A variety of monoclonal antibodies has been used to study the roles of surface proteins in neutrophil function. Many monoclonal antibodies that bind to human neutrophils react with the oligosaccharide lacto-N-fucopentaose III. Sequential immunoprecipitation of radiolabeled proteins from extracts of neutrophils labeled at the cell surface with 125I, and partial proteolysis peptide mapping studies were used to compare the proteins recognized by several widely used monoclonal antibodies that react with human neutrophils. The monoclonal antibodies that react with lacto-N-fucopentaose III (CD15) immunoprecipitated five distinct neutrophil surface proteins. The data indicate that CD15 monoclonal antibodies react with a subset of the LFA-1/HMac-1/gp 150,95 glycoprotein family as well as with CR1 on human neutrophils. The CD15 antibodies studied differed in their avidities for these proteins. The molecules immunoprecipitated by the CD15 antibodies tested were more resistant to proteolysis than the homologous proteins immunoprecipitated by the other monoclonal antibodies studied that react directly with the alpha M (CD11) or beta (CD18) chains of the LFA-1/HMac-1/gp 150,95 glycoprotein family. Some of the differences in antibody reactivity and protease sensitivity of the membrane proteins recognized by these antibodies may be due to differences in glycosylation. The data suggest that the antibodies studied can detect differences in post-translational modification among copies of certain surface proteins.     

DNA-dependent adenosinetriphosphatase A is the eukaryotic analogue of the bacteriophage T4 gene 44 protein: immunological identity of DNA replication-associated ATPases.

We report the construction of three stable murine hybridomas that secrete monoclonal antibodies which recognize calf thymus DNA-dependent adenosinetriphosphatase A. All three of the antibodies react specifically with calf thymus ATPase A and the gene 44 protein from the bacteriophage T4 DNA-dependent ATPase. Each of the three anti-ATPase A antibodies appears to recognize a different epitope and none of the antibodies inhibit DNA-dependent ATP hydrolysis by ATPase A. Furthermore, one of the antibodies has been shown to react with two different preparations of HeLa cell DNA-dependent ATPases and a yeast DNA-dependent ATPase, all of which have been implicated in the enzymology of DNA replication. These findings provide strong evidence for the role of ATPase A in DNA replication. These observations lead us to conclude that, apart from the nucleotide binding sites, there are at least three epitopes common to both the bacteriophage and eukaryotic DNA-dependent ATPases that we have examined and that the different preparations of the eukaryotic ATPases contain the same DNA-dependent ATPase.     

In situ hybridization of an acetylaminofluorene-modified probe recognized by Z-DNA antibodies in vitro

An in situ hybridization procedure, based on the chemical modification of DNA by acetylaminofluorene (AAF), followed by a specific immunoreaction was used to localize a Z-DNA sequence isolated from the satellite DNA of Cebus appella. The AAF probe is localized on the R-band-positive heterochromatic segments of Cebus chromosomes, which strongly react with Z-DNA antibodies. The use of a nonradioactive single-stranded labeled probe confirms the reliability and the rapidity of immunochemical methods for the detection of DNA sequences on chromosomes.     

The presence of natural human antibodies reactive against pharmacologically active pectic polysaccharides from herbal medicines

Direct ELISA was performed using normal human sera and human colostrum, to analyse the presence of antibodies which react with pharmacologically active pectic polysaccharides isolated from plants used in traditional Japanese herbal (Kampo) medicine. All sera and colostrum were shown to contain IgM, IgG, IgA and secretory IgA class antibodies which react with the active pectic polysaccharides to different degrees. The reacting IgG antibody in normal human serum recognized the ramified regions (rhamnogalacturonan core with carbohydrate side-chains) of the pharmacologically active pectic polysaccharides as the active sites for complement-activating activity. Correlation analysis indicated that a significant and positive correlation was observed between reactivity with the reacting antibody of IgG class and the degree of complement-activating activity of the active polysaccharides.The reacting IgG class antibody, which was purified from normal human serum by affinity chromatography on bupleuran 2IIc (a pharmacologically active pectic polysaccharide from the roots of Bupleurum falcatum)-immobilized Sepharose, showed cross-reactivity not only with some other pharmacologically active pectic polysaccharides from other medicinal herbs but also with autoantigens such as single-strand DNA, myosin and tublin from mammals.     

High levels of anti-mitochondrial antibodies in MRL mice. Influence of anti-DNA antibodies on anti-mitochondrial antibodies measured by an enzyme-linked immunosorbent assay

We developed an easy enzyme-linked immunosorbent assay (ELISA) for anti-mitochondrial antibodies, and detected high levels of anti-mitochondrial antibodies in MRL/Mp-lpr/lpr (MRL1/l) mice. The influence of the presence of anti-DNA antibodies in the tested sera on this assay was also evaluated. The monoclonal anti-DNA antibodies, which were made from non-immunized MRL1/l mice, did not react with the mitochondrial antigens adhering to polystyrene plates. Absorption with DNA had little effect on the levels of mitochondrial antibodies in MRL1/l sera. Our assay for mitochondria-binding antibody was very easy and sensitive, although it could not differentiate among heterogeneous anti-mitochondrial antibodies.     

Immunoreactivity to antinucleoside antibodies in X-irradiated HeLa cells

Synchronized HeLa cells were stained with antibodies to purine and pyrimidine nucleosides by immunofluorescent and immunoperoxidase techniques. These antibodies react only with denatured or single-stranded regions of DNA. Nuclear attachment of antibody was seen only during the period of DNA synthesis as determined by ³H-thymidine incorporation. Positive nuclear immunoreactivity was seen in approx. 15% of cells obtained by mitotic selection at a time corresponding to the G 1 phase. After exposure to ionizing radiation, 80% of the G 1 cells were reactive. Induction of immunoreactivity was dose dependent over the range of 100 to 1 000 rads. Treatment of irradiated G 1 cells with deoxyribonuclease completely eliminated the positive nuclear reaction. Exposure to ribonuclease had no effect. Incubation of the G 1 cells for 90 min at 37 or 0–4 °C after the administration of 1 000 rads resulted in a prompt decrease of immunoreactivity to control levels. However, in the presence of 0.04 μg/ml actinomycin D, positive nuclear staining remained at high levels. No such effect could be observed as a result of exposure to cytosine arabinoside or hydroxyurea. It is concluded that X-irradiation of G 1 HeLa cells produces single-stranded regions in nuclear DNA that can be detected by anti-pyrimidine and anti-purine antibodies.     

Localization of epitopes of herpes simplex virus type 1 glycoprotein D.

We previously defined eight groups of monoclonal antibodies which react with distinct epitopes of herpes simplex virus glycoprotein D (gD). One of these, group VII antibody, was shown to react with a type-common continuous epitope within residues 11 to 19 of the mature glycoprotein (residues 36 to 44 of the predicted sequence of gD). In the current investigation, we have localized the sites of binding of two additional antibody groups which recognize continuous epitopes of gD. The use of truncated forms of gD as well as computer predictions of secondary structure and hydrophilicity were instrumental in locating these epitopes and choosing synthetic peptides to mimic their reactivity. Group II antibodies, which are type common, react with an epitope within residues 268 to 287 of the mature glycoprotein (residues 293 to 312 of the predicted sequence). Group V antibodies, which are gD-1 specific, react with an epitope within residues 340 to 356 of the mature protein (residues 365 to 381 of the predicted sequence). Four additional groups of monoclonal antibodies appear to react with discontinuous epitopes of gD-1, since the reactivity of these antibodies was lost when the glycoprotein was denatured by reduction and alkylation. Truncated forms of gD were used to localize these four epitopes to the first 260 amino acids of the mature protein. Competition experiments were used to assess the relative positions of binding of various pairs of monoclonal antibodies. In several cases, when one antibody was bound, there was no interference with the binding of an antibody from another group, indicating that the epitopes were distinct. However, in other cases, there was competition, indicating that these epitopes might share some common amino acids.