Immunobiochemical characterization with monoclonal antibodies of Epstein-Barr virus-associated early antigens in chemically induced cells.

Five monoclonal antibodies which are reactive to early antigens of Epstein-Barr virus have been produced by using somatic cell hybridization techniques. The specificity of the monoclonal antibodies to early antigens was demonstrated by indirect immunofluorescence, which showed that the antigens were localized to the nucleus of early antigen-induced Raji cells. Additional indirect immunofluorescence studies showed that like patient antisera to diffuse-staining early antigen, the monoclonal antibodies gave positive staining reactions after methanol fixation. One of the antibodies, 1150-4, was positive by the anti-complement immunofluorescence technique but differed with Epstein-Barr virus-associated nuclear antigen-positive patient sera in that it only stained induced cells. Different fixation methods were found to alter dramatically the appearance of the nuclear staining reactions produced by the monoclonal antibodies. Immunoprecipitation and immunoblot experiments revealed that monoclonal antibodies 1108-1 and 1129-1 recognized two polypeptides of 55,000 and 50,000 daltons (p55;50), 1173-6 and 1180-2 recognized just p50, and 1150-4 identified a 65,000-dalton nuclear protein. Immunobiochemical characterization of these viral antigens showed that p55 is a phosphoprotein, and p55;50 has strong DNA-binding activity preferentially to single-stranded DNA. Elucidation of the role of these nuclear proteins in Epstein-Barr virus infection and the events associated with Epstein-Barr virus-directed lymphocyte transformation may provide significant information on the pathogenicity of this important human virus.     

A method for simultaneous nuclear immunofluorescence and DNA content quantitation using monoclonal antibodies and flow cytometry

A preparative technique for the two-parameter flow cytometric study of nuclear antigen expression is reported. This method employs a brief sequential treatment of cells at 4 degrees C first with 0.5% paraformaldehyde and second with 0.1% Triton X-100 in phosphate-buffered saline followed by cellular staining with indirect immunofluorescence and propidium iodide. Using this technique, cellular morphology is preserved, cell clumping is minimized, and high-quality indirect immunofluorescence and DNA staining are obtained with a minimum of nonspecific labeling. Utilizing nuclear antigen-specific monoclonal antibodies in conjunction with this technique, the cell-cycle phase-dependent expression of such antigens is examined. From these data, the utility of two-parameter flow cytometry in the identification and quantification of cell-cycle-dependent modulation of nuclear antigens is discussed.     

Light and electron microscopic immunolocalization of two nuclear antigens in the liver of Pleurodeles waltl using monoclonal antibodies

The distribution of 2 nuclear antigens in the interphase nuclei of liver of Pleurodeles waltl was determined with the help of monoclonal antibodies, using immunofluorescence for light microscopy and indirect immunoperoxidase and immunogold labeling procedures for electron microscopic localization. The antibodies C36/1 and A33/22 label antigens with relative molecular masses of 270 kDa and 80 kDa, and isoelectric points of 7.0 and 6.4, respectively. The liver of urodels is characterized by the presence of a peripheral layer of hematopoietic cells around the parenchymatous tissue formed by typical hepatocytes. The antibody C36/1 labels the nuclei of both types of cells, whereas the antibody A33/22 labels the nuclei of hepatocytes but not those of the peripheral hematopoietic cells. With both these antibodies, labeling, whenever observed, is restricted to fibrillar structures in the interchromatin space, i.e., to peri- and inter-chromatin fibrils; condensed chromatin, nucleoli, and nuclear envelope are not labeled.     

Monoclonal antibodies identify a group of nuclear pore complex glycoproteins

Using monoclonal antibodies we identified a group of eight polypeptides of rat liver nuclear envelopes that have common epitopes. Most or all of these proteins are structurally distinct, as shown by tryptic peptide mapping and analysis with polyclonal antibodies. While these polypeptides are relatively tightly bound to nuclear membranes, only one is an integral membrane protein. The eight antigens cofractionate with the nuclear pore complex under various conditions of ionic strength and detergent. It can be seen by immunofluorescence microscopy that the monoclonal antibodies reacting with these antigens stain the nuclear surface of interphase cells in a finely punctate pattern. When the nuclear envelope is disassembled and subsequently reformed during mitosis, the proteins are reversibly dispersed throughout the cytoplasm in the form of minute foci. By EM immunogold localization on isolated nuclear envelopes, the monoclonal antibodies label exclusively the nuclear pore complex, at both its nucleoplasmic and cytoplasmic margins. Considered together, our biochemical and localization data indicate that the eight nuclear envelope polypeptides are pore complex components. As shown in the accompanying paper (Holt, G. D., C. M. Snow, A. Senior, R. S. Haltiwanger, L. Gerace, and G. W. Hart, J. Cell Biol., 104:1157-1164) these eight polypeptides contain a novel form of glycosylation, O-linked N-acetylglucosamine. The relative abundance and disposition of these O-linked glycoproteins in the pore complex are consistent with their having a role in nucleocytoplasmic transport.     

Identification of a nuclear protein component of interchromatin granules using a monoclonal antibody and immunogold electron microscopy

A monoclonal antibody, designated 780-3, has been generated which preferentially recognizes an antigenic component of interchromatin granules in human cells. By indirect immunofluorescence procedures, monoclonal antibody 780-3 produces a cell cycle-specific speckled nuclear staining pattern in adult human fibroblasts which is dramatically altered during metaphase. In contrast, transformed cells appear to express this antigen throughout the cell cycle in increased quantities. Immunogold electron microscopy revealed that the nuclear antigen is intimately associated with interchromatin granules in human cells. Analysis by immunoblot procedures showed that monoclonal antibody 780-3 recognizes two polypeptides of 105 and 41 kD. From these data, a possible nucleolar derivation of interchromatin granules is discussed. These studies demonstrate for the first time that monoclonal antibodies may be used in combination with immunogold electron microscopy to identify the ultrastructural location of nuclear antigens.     

A library of monoclonal antibodies to nuclear proteins from Drosophila melanogaster embryos. Characterization by a cultured cell assay

To study the structure and function of the cell nucleus, a library of 170 monoclonal antibodies was produced to nuclear antigens from 3-6 h old Drosophila embryos. In preparation for immunization, nuclei were separated, at neutral pH and in the presence of polyamines, into two fractions containing either urea-soluble non-histone nuclear proteins or histones plus small quantities of non-histone proteins complexed to DNA. The antibodies were characterized in a rapid, indirect immunofluorescent assay employing cultured Drosophila cells (Schneider's line 2). Low backgrounds and high specific fluorescence were achieved in this assay by purifying the rhodamine-labelled second antibody on a polystyrene resin and washing the cells with optimal concentrations of detergents. The assay categorized antigens according to their cellular locations: in nuclei, in nuclei plus cytoplasm, or primarily in cytoplasm. A subset of nuclear antigens reacted specifically with the nuclear envelope. In addition, some antibodies were characterized by their reactions with polytene chromosomes. The cultured cell assay provides a new, efficient method for expanding this antibody library. The monoclonal antibodies in the library now provide highly specific tools for investigating structural nuclear proteins and proteins that may be regulatory during embryonic development.     

Localization of cyclic AMP-dependent protein kinase subunits in rat hepatocyte nuclei by immunogold electron microscopy

We have applied the indirect colloidal immunogold technique to examine the ultrastructural localization of the catalytic subunit C and the regulatory subunits RI and RII of cyclic AMP-dependent protein kinase in rat hepatocyte nuclei before and after glucagon or dibutyryl cyclic AMP administration. The technique allowed the identification and localization of all three subunits in hepatocyte nuclei. Morphometric quantitation of the relative staining density of nuclear subunits indicated an increase of immunogold staining of nuclear catalytic subunit but not of the regulatory subunits after glucagon or dibutyryl cyclic AMP stimulation. The increase of catalytic subunit occurred in a biphasic manner with peak levels 2-30 min and 90-150 min after stimulation. Our experiments represent the first reported use of the immunogold procedure to identify and localize protein kinase subunits in the nucleus.     

Application of denatured, electrophoretically separated, and immobilized lysates of herpes simplex virus-infected cells for detection of monoclonal antibodies and for studies of the properties of viral proteins

We report the use of herpes simplex virus type 1 (HSV-1)- and HSV-2-infected cell polypeptides (ICPs) separated by electrophoresis in polyacrylamide gels and transferred to nitrocellulose to (i) detect monoclonal antibodies to viral polypeptides and to (ii) study the properties of the proteins with the monoclonal antibodies. Our results were as follows. (i) When the antigens were electrophoretically separated in denaturing gels and then immobilized on nitrocellulose strips, we detected a greater diversity of monoclonal antibodies to viral proteins than when we used the technique of immune precipitation of soluble, nondenatured viral antigens. The primary advantage of the technique is in the detection of nonprecipitating antibody and of antibody to poorly soluble antigens not available for reaction in preparations cleared by high-speed centrifugation before immune reaction. (ii) Studies of the viral polypeptides reactive with three monoclonal antibodies indicated that the technique can be used to investigate several properties of the antigens. Specifically, monoclonal antibody to ICP 4 confirmed the accumulation of viral protein in the nucleus and the mapping of the gene in the S component. The results showed, however, that HSV-1 and HSV-2 ICP 4 do have common antigenic determinants. The reaction of a nonprecipitating monoclonal antibody with electrophoretically separated, immobilized polypeptides contained in cytoplasmic and nuclear fractions, those chemically deglycosylated, or those specified by specific HSV-1 x HSV-2 intertypic recombinants identified the antigens reactive with the second monoclonal antibody as various forms of glycoprotein gC. Of particular interest was a set of four antigens, 39,000 to 46,500 in apparent molecular weight, reactive with each of several monoclonal antibodies. These studies showed that two polypeptides partition in the cytoplasm and two in the nucleus and that all comap with the previously mapped ICPs 35 and 37 in the region of the genome defined by the viral thymidine kinase gene on the left and the glycoprotein gA/B gene on the right. Unlike ICP 4 and gC, the four polypeptides are linked by intermolecular bisulfide bonds, inasmuch as the polypeptides were not at the expected locations upon denaturation and electrophoresis in the absence of reducing agents.     

Localization of nuclear subunits of cyclic AMP-dependent protein kinase by the immunocolloidal gold method

An immunocolloidal gold electron microscopy method is described allowing the ultrastructural localization and quantitation of the regulatory subunits RI and RII and the catalytic subunit C of cAMP-dependent protein kinase. Using a postembedding indirect immunogold labeling procedure that employs specific antisera, the catalytic and regulatory subunits were localized in electron-dense regions of the nucleus and in cytoplasmic areas with a minimum of nonspecific staining. Antigenic domains were localized in regions of the heterochromatin, nucleolus, interchromatin granules, and in the endoplasmic reticulum of different cell types, such as rat hepatocytes, ovarian granulosa cells, and spermatogonia, as well as cultured H4IIE hepatoma cells. Morphometric quantitation of the relative staining density of nuclear antigens indicated a marked modulation of the number of subunits per unit area under various physiologic conditions. For instance, following partial hepatectomy in rats, the staining density of the nuclear RI and C subunits was markedly increased 16 h after surgery. Glucagon treatment of rats increased the staining density of only the nuclear catalytic subunit. Dibutyryl cAMP treatment of H4IIE hepatoma cells led to a marked increase in the nuclear staining density of all three subunits of cAMP-dependent protein kinase. These studies demonstrate that specific antisera against cAMP-dependent protein kinase subunits may be used in combination with immunogold electron microscopy to identify the ultrastructural location of the subunits and to provide a semi-quantitative estimate of their relative cellular density.     

Changes in distribution of nuclear matrix antigens during the mitotic cell cycle

We examined the distribution of nonlamin nuclear matrix antigens during the mitotic cell cycle in mouse 3T3 fibroblasts. Four monoclonal antibodies produced against isolated nuclear matrices were used to characterize antigens by the immunoblotting of isolated nuclear matrix preparations, and were used to localize the antigens by indirect immunofluorescence. For comparison, lamins and histones were localized using human autoimmune antibodies. At interphase, the monoclonal antibodies recognized non-nucleolar and nonheterochromatin nuclear components. Antibody P1 stained the nuclear periphery homogeneously, with some small invaginations toward the interior of the nucleus. Antibody I1 detected an antigen distributed as fine granules throughout the nuclear interior. Monoclonals PI1 and PI2 stained both the nuclear periphery and interior, with some characteristic differences. During mitosis, P1 and I1 were chromosome-associated, whereas PI1 and PI2 dispersed in the cytoplasm. Antibody P1 heavily stained the periphery of the chromosome mass, and we suggest that the antigen may play a role in maintaining interphase and mitotic chromosome order. With antibody I1, bright granules were distributed along the chromosomes and there was also some diffuse internal staining. The antigen to I1 may be involved in chromatin/chromosome higher-order organization throughout the cell cycle. Antibodies PI1 and PI2 were redistributed independently during prophase, and dispersed into the cytoplasm during prometaphase. Antibody PI2 also detected antigen associated with the spindle poles.     

Immunogold electron microscopy of surface antigens of oral bacteria

Colloidal gold particles 3-6 nm in diameter were prepared and stabilized with the IgG fraction of polyspecific rabbit antisera produced against four different oral bacteria. The immunogold markers were used in homologous reactions to label the bacteria in a preembedding procedure for electron microscopy. An indirect immunofluorescence procedure was concurrently used to optimize the labelling conditions before observation with the electron microscope. The immunogold markers labelled fibrillar structures extending outward 50-275 nm from the Gram-positive cell envelopes and a fuzzy 5-10 nm thick capsulelike layer on the outer aspect of Bacteroides gingivalis. The immunogold method appears to be a simple, rapid, and inexpensive procedure suitable for the study of bacterial surface antigens and can be upgraded with the use of monospecific antibodies.     

Electron microscopic localization of glutamate dehydrogenase in rat liver mitochondria by an immunogold procedure and monoclonal and polyclonal antibodies

Glutamate dehydrogenase (GDH) was localized in rat liver by indirect electron microscopic immunogold, using different sizes of gold particles and monoclonal and polyclonal antibodies. Using the protein A-gold technique in double immunocytochemical experiments, both antibodies, at their optimal dilutions, gave similar results. A novel assessment of the distribution of GDH was made by measurements of the number of gold particles per square micrometer of cross-sectional images of individual mitochondria. The data indicate intracellular homogeneity among mitochondria in individual parenchymal cells. The enzyme is almost absent in non-parenchymal cells. Finally, GDH was found mainly in association with the mitochondrial inner membrane.     

Generation of monoclonal antibodies against chromosomal antigens that have a high sequence similarity between human and mouse

We raised monoclonal antibodies by immunizing mice with total chromosome proteins extracted from isolated human metaphase chromosomes. The indirect immunofluorescence screening of hybridoma cell lines provided 15 monoclonal antibodies against the chromosomal antigens. The antigen proteins of the mAbs were identified by immunoblotting as core histones or by immunoprecipitation followed by a peptide mass fingerprinting method as nuclear mitotic apparatus protein, heterogeneous nuclear ribonucleoprotein A2/B1, ribosomal protein S4, linker histone and beta-actin. During mitosis, localizations of these proteins on chromosomes were clearly observed using the obtained antibodies. These results indicate that the current strategy is effective for obtaining monoclonal antibodies useful for immunoblotting and/or immunofluorescent staining of human proteins, using the antigens with high homology to mouse proteins.     

Co-localization of non-histone protein BA with U-snRNPs to the same regions of the cell nucleus

Using indirect immunofluorescence, nuclear non-histone protein BA was localized in a normal rat liver cell line. Protein BA antibodies immunostained nuclear structures producing a speckled immunofluorescent staining pattern. Nuclear structures stained with protein BA antibodies were sensitive to DNase I digestion, but not to RNase. The speckled pattern of nuclear fluorescence observed with protein BA antibodies was similar to that reported earlier for Sm antibodies, which react with U-snRNPs. Using double-label indirect immunofluorescence, the Sm antigen was shown to be concentrated in the same regions of the nucleus which contain protein BA. Immunoblot analysis of total nuclear proteins with the two antibodies demonstrated that protein BA and the major Sm antigen have similar molecular weights, but are antigenically distinct. In addition, they differ in their extractabilities from the cell nucleus.     

Identification of two major components of the lateral elements of synaptonemal complexes of the rat

This paper describes the identification of two major components of the lateral elements of synaptonemal complexes of the rat by immunocytochemical techniques. We prepared monoclonal antibodies against synaptonemal complexes (SCs) by immunization of mice with purified SCs. One of these antibodies, II52F10, reacts with a 30 and a 33 kDa polypeptide, which are major components of purified SCs. Using this antibody, we studied the localization of its antigens light microscopically, by means of the indirect immunoperoxidase technique, as well as ultrastructurally, by means of the immunogold labeling technique. The immunolocalization was carried out on whole-mount preparations of lysed spermatocytes. The antibody reacts with paired as well as unpaired segments of zygotene, pachytene and diplotene SCs. In light microscopic preparations, the attachment plaques, particularly those of late pachytene and diplotene SCs, also appear to react strongly. In electron micrographs the lateral elements in paired as well as unpaired segments could be seen to react. No reaction was observed in the attachment plaques; however, in late pachytene and diplotene SCs the swollen terminal segments of the lateral elements did react with the antibody. Thus, we conclude that a 30 and a 33 kDa polypeptide make part of the lateral elements of synaptonemal complexes of the rat.     

Quantitative Changes in Periplasmic Proteins of the Macronucleus-Specific Bacterium Holospora obtusa in the Infection Process of the Ciliate Paramecium caudatum

ABSTRACT. The Gram-negative bacterium Holospora obrusa is a macronucleus-specific symbiont of the ciliate Paramecium caudatum. The infectious form of this bacterium infects the host macronucleus through digestive vacuoles and differentiates into the reproductive form two days after the infection in the nucleus. The monoclonal antibodies IF-3–1 and IF-3–2 reacted with 39 and 1S kDa periplasmic proteins, respectively, that were specific for the infectious form of H. obrusa. Because the antigens were not detected in the reproductive form of the bacterium, it appears that expression of the proteins decreases during or soon after the infection. Using these antibodies, quantitative changes in the antigens in the early infection process were examined by immunoblotting and immunogold electron microscopy. Immunoblotting showed that the amounts of both antigens were reduced within 1 h after the bacteria were engulfed into the digestive vacuoles of the paramecia, but that the amounts of IF-3–2 antigens declined earlier than the IF-3–1 antigen. Immunogold labeling showed that the level of IF-3–2 antigens became very low in the bacteria in the host digestive vacuoles, whereas there was no similar decrease in amount of IF-3–1 antigens. Possible functions of the antigens are discussed. The IF-3–1 antigens decrease in concentration in parallel with the decrease in the periplasmic region.     

Ultrastructural localization of the circulating anodic antigen and the circulating cathodic antigen in the liver of mice infected with Schistosoma mansoni: a sequential study

The present study describes the ultrastructural localization of two important circulating schistosome antigens--the circulating anodic antigen (CAA) and the circulating cathodic antigen (CCA)--in livers of mice at various time intervals after infection with Schistosoma mansoni. For the demonstration of these antigens at the electron microscope level use was made of a direct, double immunogold labeling procedure, in which CAA-specific monoclonal antibodies, labeled with 5-nm gold particles, and CCA-specific monoclonal antibodies, labeled with 15-nm gold particles, were used. Both antigens were localized in granules and in inclusion bodies of Kupffer cells and granuloma macrophages and it was found that in these compartments the degree of 5- and 15-nm gold labeling increased with the duration of the infection. Sometimes gold particles were also encountered on the cell surface and in endocytotic vesicles of these cells, in endothelial cells, and in the space of Disse. From these data it was concluded that in the liver CAA and CCA were primarily accumulated in granules and inclusion bodies of Kupffer cells and granuloma macrophages. It is discussed whether at these locations both antigens are degraded by lysosomal enzymes and whether these antigens are complexed with antibodies.     

A cell surface antigen associated with meiosis in male Staurdoderus scalaris (Orthopteran).

Immunofluorescence has identified seven monoclonal antibodies reactive with the surface of meiotic cells and absent in premeiotic cells. Analysis by immunogold electron microscopy indicated that these antigens were present on the external surface of the cells and were coincident with the presence of synaptonemal complexes in the nucleus. On immunoblots a common glycosylated protein of 205 kDa was recognized, in addition to smaller subunits, suggesting the presence of a protein complex comprised of smaller peptides.     

Monoclonal antibody specific for human nuclear proteins IEF 8Z30 and 8Z31 accumulates in the nucleus a few hours after cytoplasmic microinjection of cells expressing these proteins

A monoclonal antibody (mAB 1C4C10) that reacts specifically with human nuclear proteins IEF 8Z30 and 8Z31 (charge variants; HeLa protein catalogue number; Bravo, R., and J. E. Celis, 1982, Clin. Chem., 28:766- 781) has been microinjected into the cytoplasm of cultured cells that either express (primates) or lack these proteins (at least having similar molecular weights and pIs; other species), and its cellular localization has been determined by indirect immunofluorescence. Nuclear localization (nucleolar and nucleoplasmic) of the antibody was observed only in cells expressing these antigens, suggesting that a determinant present in IEF 8Z30 and 8Z31 is required for cytoplasm- nuclear translocation. Nuclear migration was not inhibited by cycloheximide, implying that these proteins may shuttle between nucleus and cytoplasm. The results assumed to support the signal rather than the free diffusion model are further supported by microinjection experiments using antibodies (proliferating cell nuclear antigen/cyclin, DNA) that react with nuclear components but do not recognize cytoplasmic antigens. Furthermore, they raise the possibility that some nonnuclear proteins may be transported to the nucleus by interacting with proteins harboring nuclear location signals.