Studies of murine malaria antigens using monoclonal antibodies. Production, selection, and characterization of antibodies

A panel of ten monoclonal antibodies made against Plasmodium chabaudi and Plasmodium yoelii infected mouse erythrocytes were used for characterization of antigens present in murine malaria. Screening of the antibodies in ELISA with different fractions of infected erythrocytes revealed both species-specific and fraction-specific monoclonal antibodies (MAbs), but also MAbs cross-reacting between the species. Two MAbs bound normal erythrocyte components. Subcellular localization of the target antigens was studied by immunofluorescence and their molecular identity by immunoblotting after SDS-PAGE. Of the MAbs to P. yoelii, one reacted with a cytoplasmic granule component of 137 k and two others reacted with vacuole-associated antigens of 26 k and 25/70/73 k, respectively. The latter antibodies cross-reacted with P. chabaudi antigens. Of the MAbs to P. chabaudi, all were species specific, one reacting with parasite surface antigens of 79 and 250 k and two with a vacuole-associated antigen of 70 k.     

Human liver catalase: cloning,expression and characterization of monoclonal antibodies

We isolated a cDNA encoding liver catalase from a human liver cDNA library. The cDNA had a high degree of sequence similarity to the corresponding enzyme from other sources. It was expressed in E. coli using the pET15b vector. The protein produced was enzymatically active after purification, and its kinetic parameters closely resembled those of other mammalian catalases. Monoclonal antibodies were generated against the purified catalase; six antibodies recognizing different epitopes were obtained, one of which inhibited the enzyme. The cross reactions of the antibodies with brain catalases from human and other mammalian tissues were investigated, and all the immunoreactive bands obtained on Western blots had molecular masses of about 58 kDa. Similarly fractionated extracts of several mammalian cell lines all gave a single band of molecular mass 58 kDa. These results indicate that mammalian livers and human cell lines contain only one major type of immunologically reactive catalase, even though some of catalases have been previously reported to differ in certain properties.     

The effect of taxol onTriticum preprophase root cells: preprophase microtubule band organization seems to depend on new microtubule assembly

Summary To examine whether preprophase microtubule band (PPB) organization occurs by rearrangement of pre-existing, or by assembly of new microtubules (Mts), we treated root cells ofTriticum turgidum with taxol, which stabilizes pre-existing Mts by slowing their depolymerization. With taxol early preprophase cells failed to form a normal PPB and PPB narrowing was prevented in cells that had already formed a wide one. The PPB became persistent in prometaphase cells and the formation of multipolar prophase-prometaphase spindles was induced. These data favour the suggestion that PPB formation and narrowing, as well as prophase spindle development, are dynamic processes depending on continuous Mt assembly at the PPB site and in the perinuclear cytoplasm.Abbreviations Mt microtubule - MTOC microtubule organizing centre - PPB preprophase microtubule band - DMSO dimethyl sulfoxide     

Human mononuclear phagocyte-associated antigens: I. Identification and characterization

Rabbit antisera were produced against a lymphokine-activated human macrophage cell line, U937 (αU937), and human peritoneal macrophages (αPEMØ). After absorption with AB erythrocytes, pooled platelets, and B-lymphoblastoid cell lines, both antisera reacted by microcytotoxicity, indirect immunofluorescence (IF), and radioimmunoassay (RIA) with adherence-purified human peripheral blood monocytes, splenic and peritoneal macrophages, and leukemic myelomonoblasts. A panel of normal human T lymphocytes, B lymphocytes, and erythroid-myeloid or lymphoblastoid cell lines failed to react with both αU937 and αPEMØ. Although both heteroantisera reacted against polymorphonuclear leukocytes (PMNs), after absorption with PMNs specific reactivity against mononuclear phagocytes remained. Absorption of αU937 and αPEMØ with myelomonoblastic leukemia cells (AMML) removed IF and RIA activity against both PMNs and monocytes but not against splenic and peritoneal macrophages. In contrast, absorptions of both heteroantisera preparations with splenic macrophages abolished their IF and RIA reactivity not only to splenic and peritoneal macrophages but also to peripheral blood monocytes and leukemic myelomonoblasts. These results are consistent with (1) both antisera defining specific monocyte/macrophage-associated antigens(s) which are distinct from MHC-coded HLA-A,B,C, and DR antigens, and (2) expression of common monocyte/macrophage-associated antigen(s) and uniquely associated antigen(s) selectively expressed on tissue macrophages. These reagents will be useful in delineating human monocyte/macrophage differentiation as well as the immunological functions of mononuclear phagocytes.     

An immunofluorescent study of the microtubule organization in Trichomonas vaginalis using antitubulin antibodies

The flagellated protozoon Trichomonas vaginalis, parasite of the human urogenital tract, possesses a well developed microtubule system organized in highly differentiated structures. We have shown by immunoblotting that monospecific anti-sheep brain tubulin antibodies are able to react with the microtubular tubulin of T. vaginalis. These antibodies were used to study the microtubular system of T. vaginalis both in interphase and mitosis by indirect immunofluorescence. The interphase microtubular pattern, characterized by an axostyle, a pelta, four anterior flagella, and a recurrent flagellum, displayed remarkable changes at the onset of mitosis: the axostyle disappeared, and two pole bodies connected by a short spindle became evident; chromosomal fibers arose while pole-to-pole fibers elongated. The last phases of mitosis were marked by the disappearance of chromosomal fibers, the appearance of two small axostyles, and the depolymerization of the pole-to-pole bundle. At the end of mitosis, the normal interphase microtubule pattern was observed.     

Myoseverin disrupts sarcomeric organization in myocytes: an effect independent of microtubule assembly inhibition

Although disruption of the microtubule (MT) array inhibits myogenesis in myocytes, the relationship between the assembly of microtubules (MT) and the organization of the contractile filaments is not clearly defined. We now report that the assembly of mature myofibrils in hypertrophic cardiac myocytes is disrupted by myoseverin, a compound previously shown to perturb the MT array in skeletal muscle cells. Myoseverin treated cardiac myocytes showed disruptions of the striated Z-bands containing alpha-actinin and desmin and the localization of tropomyosin, titin and myosin on mature sarcomeric filaments. In contrast, MT depolymerization by nocodazole did not perturb sarcomeric filaments. Similarly, expression of constitutively active stathmin as a non-chemical molecular method of MT depolymerization did not prevent sarcomere assembly. The extent of MT destabilization by myoseverin and nocodazole were comparable. Thus, the effect of myoseverin on sarcomere assembly was independent of its capacity for MT inhibition. Furthermore, we found that upon removal of myoseverin, sarcomeres reformed in the absence of an intact MT network. Sarcomere formation in cardiac myocytes therefore, does not appear to require an intact MT network and thus we conclude that a functional MT array appears to be dispensable for myofibrillogenesis.     

Production and characterization of monoclonal antibodies to Aeromanas sobria surface antigens

Abstract A panel of eight murine monoclonal antibodies (Mabs) was raised against the surface antigens of Aeromonas sobria isolated from a patient with diarrhoea. Antibodies to the LPS core and O-antigen and to protein antigens were generated. Three Mabs of the IgM isotype against either protein or LPS agglutinated 34/75 Aeromonas isolates from clinical and environmental sources. Similar numbers of A. hydrophila, A. sobria and A. caviae isolates were agglutinated. The Mabs were screened for their ability to inhibit A. sobria adhesion to HEp-2 cells, and haemagglutination (HA). Two Mabs directed against conformationally dependent epitopes on a 43-kDa protein blocked both functions. Of the anti-LPS Mabs one blocked adhesion only, and another blocked HA but not adhesion. Immunoprecipitation studies suggested that LPS-protein complexes may be involved in these potential virulence functions of A. sobria .     

Production and characterization of monoclonal antibodies to Aeromonas sobria surface antigens

A panel of eight murine monoclonal antibodies (Mabs) was raised against the surface antigens of Aeromonas sobria isolated from a patient with diarrhoea. Antibodies to the LPS core and O-antigen and to protein antigens were generated. Three Mabs of the IgM isotype against either protein or LPS agglutinated 34/75 Aeromonas isolates from clinical and environmental sources. Similar numbers of A. hydrophila, A. sobria and A. caviae isolates were agglutinated. The Mabs were screened for their ability to inhibit A. sobria adhesion to HEp-2 cells, and haemagglutination (HA). Two Mabs directed against conformationally dependent epitopes on a 43-kDa protein blocked both functions. Of the anti-LPS Mabs one blocked adhesion only, and another blocked HA but not adhesion. Immunoprecipitation studies suggested that LPS-protein complexes may be involved in these potential virulence functions of A. sobria.     

The effect of okadaic acid on Arabidopsis thaliana root morphology and microtubule organization in its cells

To investigate the role of protein hyperphosphorylation in plant cells, the effect of okadaic acid, a specific inhibitor of protein phosphatases PPI and PP2A, on the general morphology of Arabidopsis thaliana primary roots and the structural-functional characteristics of cortical microtubules in different cell types in all primary root growth zones was studied. It was found that okadaic acid affects microtubule organization in a different manner depending on the type of cells and functional zones of the primary root. Cortical microtubules in the epidermis and cortex cells of the elongation zone proved to be most sensitive to 0.1, 1, and 10 nM okadaic acid which completely depolymerized after inhibitor treatment. In trichoblasts, atrichoblasts of differentiation zone treatment with okadaic acid caused the microtubules stabilization. The treatment with okadaic acid significantly affected the morphology of root hairs, causing their swelling and branching as a result of abnormal microtubule orientation. The results of this study suggest that induction of protein hyperphosphorylation as a result of protein phosphatase inhibition plays a crucial key in microtubule organization in plant cells.     

Islet cell antigens and autoantigen(s): Monoclonal antibodies and further characterization

A novel series of murine monoclonal antibodies to islet cells (1–45, 1–51, 1–52 and 1–39) have been generated using human insulinoma homogenate as the immunogen in order to characterize pathogenetically relevant islet cell autoantigen(s). Differentiation antigens recognized by these islet cell monoclonal antibodies displayed varied cytological distribution (pan-islet or peripheral mantle only). Monoclonal antibody 1–45 reacted with all endocrine subsets of the pancreatic islet, similar to the reactivity of islet cell autoantibody positive sera from type I diabetes subjects. Preexposure to pH2 abolished the immunoreactivity of the autoantigen; 1–45 antigen was also sensitive to low pH. Preexposure to 100° C for 1 h did not significantly alter the immunoreactivity of islet antigens recognized by ICAb positive patient sera and monoclonal antibody 1–39, thus demonstrating the extraordinary heat stability of the corresponding epitopes; those recognized by 1–45 were less heat stable. Islet cells were found to share 1–45 differentiation antigen(s)/epitope(s) with other neuroendocrine cells,viz. amerior pituitary, adrenal medulla and gut endocrine cells.     

HLA-A,B antigens Ia-like antigens, and tumor-associated antigens on prostate carcinoma cell lines: serologic and immunochemical analysis with monoclonal antibodies

Serologic and immunochemical analysis of the antigenic profile of the 2 human prostate carcinoma cell lines DU-145 and H494 with a battery of monoclonal antibodies has shown that both cell lines express HLA-A,B alloantigens and the 94,000 m.w. tumor-associated glycoprotein recognized by the monoclonal antibody 376.96S. In addition, the cell line H494 unexpectedly expresses Ia-like antigens, which are similar in their antigenic profile and structure to B lymphoid cell derived Ia-like antigens. Both Ia-like antigens and tumor-associated antigens can function as targets of cell-dependent lysis mediated by the corresponding monoclonal antibodies.     

Human specific anti-type IV collagen monoclonal antibodies,characterization and immunohistochemical application

Summary This paper describes two new monoclonal antibodies reactive with human specific type IV collagen epitopes in frozen as well as routinely fixed and processed tissue sections. The antibodies (1042 and 1043) were raised against human placental type IV collagen and were shown by immunoblotting and ELISA tests to react exclusively with type IV collagen determinants. Extensive immunohistochemical survey studies on panels of tissues from various species, using unfixed cryosat sections, demonstrated that antibody 1042 reacted only with human type IV collagen whereas antibody 1043 in addition reacted with rabbit type IV collagen. All tissues showed homogeneous staining of the basement membrane, indicating that the detected epitopes did not show organ-specific distribution.Tissue processing protocols for using these monoclonal antibodies on routinely processed paraffin embedded tissues were developed. It was found that whereas polyclonal antitype IV collagen antisera required pepsin digestion, our monoclonal antibodies required pronase or papain digestion to restore type IV collagen immunoreactivity in paraffin sections.It is concluded that these monoclonal anti-type IV collagen antibodies detect species specific epitopes which can be detected in routinely processed paraffin embedded tissues after appropriate enzyme pretreatment.     

Human specific anti-type IV collagen monoclonal antibodies, characterization and immunohistochemical application

This paper describes two new monoclonal antibodies reactive with human specific type IV collagen epitopes in frozen as well as routinely fixed and processed tissue sections. The antibodies (1042 and 1043) were raised against human placental type IV collagen and were shown by immunoblotting and ELISA tests to react exclusively with type IV collagen determinants. Extensive immunohistochemical survey studies on panels of tissues from various species, using unfixed cryostat sections, demonstrated that antibody 1042 reacted only with human type IV collagen whereas antibody 1043 in addition reacted with rabbit type IV collagen. All tissues showed homogeneous staining of the basement membrane, indicating that the detected epitopes did not show organ-specific distribution. Tissue processing protocols for using these monoclonal antibodies on routinely processed paraffin embedded tissues were developed. It was found that whereas polyclonal anti-type IV collage antisera required pepsin digestion, our monoclonal antibodies required pronase or papain digestion to restore type IV collagen immunoreactivity in paraffin sections. It is concluded that these monoclonal anti-type IV collagen antibodies detect species specific epitopes which can be detected in routinely processed paraffin embedded tissues after appropriate enzyme pretreatment.     

Tumor-associated glycolipid antigens, their metabolism and organization

A number of experimental animal tumors as well as human cancers have been characterized by dramatic changes of glycolipid composition and metabolism. This review focuses on the chemical and enzymatic basis of the appearance of tumor-associated glycolipid antigens belonging to four major structural classes, i.e., globo, ganglio, lacto type 1, and lacto type 2 series. Some antigens represent the accumulation of precursors with deletion of more complex glycolipids, and others are the result of enhanced synthesis of new structures, most of which are aberrant fucosylation or sialylation or their combination; thus, novel structures such as di- or trimeric Le chi, trifucosyl Le gamma, sialyl Le chi, sialyl dimeric Le chi and disialyl Le alpha A have been isolated and characterized. Many monoclonal antibodies are capable of recognizing antigens in high density but are not capable of reacting with the same antigen in low density. Therefore, the expression of novel structures in high densities at the cell surface is important for recognition of tumor-association antigens. Molecular models of a typical tumor-associated antigen and its organization in membranes are also presented.     

A rat monoclonal antibody reacting specifically with the tyrosylated form of alpha-tubulin. I. Biochemical characterization, effects on microtubule polymerization in vitro, and microtubule polymerization and organization in vivo

The antigenic site recognized by a rat monoclonal antibody (clone YL 1/2) reacting with alpha-tubulin (Kilmartin, J.V., B. Wright, and C. Milstein, 1982, J. Cell Biol., 93:576-582) has been determined and partially characterized. YL 1/2 reacts specifically with the tyrosylated form of brain alpha-tubulin from different mammalian species. YL 1/2 reacts with the synthetic peptide Gly-(Glu)3-Gly-(Glu)2- Tyr, corresponding to the carboxyterminal amino acid sequence of tyrosylated alpha-tubulin, but does not react with Gly-(Glu)3-Gly- (Glu)2, the constituent peptide of detyrosylated alpha-tubulin. Electron microscopy as well as direct and indirect immunofluorescence microscopy shows that YL 1/2 binds to the surface of microtubules polymerized in vitro and in vivo. Further in vitro studies show that the antibody has no effect on the rate and extent of microtubule polymerization, the stability of microtubules, and the incorporation of the microtubule-associated proteins (MAP2) and tau into microtubules. In vivo studies using Swiss 3T3 fibroblasts injected with YL 1/2 show that; when injected at low concentration (2 mg IgG/ml in the injection solution), the antibody binds to microtubules without changing their distribution in the cytoplasm. Injection of larger concentration of YL 1/2 (6 mg IgG/ml) induces the formation of microtubule bundles, and still higher concentrations cause the aggregation of microtubule bundles around the nucleus (greater than 12 mg IgG/ml).     

Isolation of monoclonal antibodies to chromatin and preliminary characterization of target antigens

This paper describes the isolation of monoclonal antibodies to chromatin-associated protein antigens and their use in the characterization of such proteins by indirect immunofluorescence. Hybridomas were derived by fusion of the mouse myeloma Ag8653 with spleen cells from mice immunized with chromatin from human liver, rat liver or a human lymphoblastoid cell line. Hybrids were screened by solid-phase radioimmunoassay. The proportion of positive hybrids varied with the immunizing chromatin as follows: human liver 55/83, human lymphoblast 8/183 and rat liver 2/82. Fifteen antibodies derived from these fusions (7, 7 and 1 respectively) were subjected to further analysis. Most of these (11/13) were IgM and recognized both human and rat chromatin (12/15). Most of the target antigens were protease sensitive (8/13) and nuclease resistant. In fact the binding of five antibodies to lymphoblast chromatin was more than doubled by preincubation with DNAase I. The subcellular location of target antigens was examined by indirect immunofluorescence. Seven antibodies stained at least one of several cultured cell lines tested. Three gave staining patterns consistent with the in vivo association of the target antigen with chromatin recognizing, respectively, the interphase nucleus and metaphase chromosomes, the nuclear periphery and the mitotic spindle and other microtubule-containing structures. The remaining four all recognized antigens associated with the intermediate filament network.     

Cell-cell signalling,microtubule organization and RNA localization: Is PKA a link?

Specification of the anterior-posterior axis of the Drosophila embryo is brought about by the asymmetric localization of specific maternally expressed RNAs and proteins within the oocyte. While many of these localized molecules have been identified and progress has been made towards understanding their functions, how the localization process is instigated remains unclear. A recent paper reports that protein kinase A (PKA) activity is essential for many of these RNA localizations and for the correct polarization of the microtubule cytoskeleton⁽¹⁾. These and other results support a model for anterior-posterior axis establishment which involves intercellular signalling between the oocyte and certain neighbouring somatic cells.     

Human polyreactive and monoreactive antibodies: effect of glycosylation antigen binding

The present experiments were initiated to determine whetherthe carbohydrate portions of antibody molecules contribute topolyreactivity. Cell lines making human monoclonal polyreactiveor monoreactive antibodies of the immunoglobulin (Ig) M, IgGand IgA isotypes were treated with tunicamycin to block N-linkedglycosylation of the proteins. Analysis of the secreted nativeand non-glycosylated proteins revealed a >95% inhibitionof [³H]mannose incorporation. Electrophoresis on sodium dodecylsulphate-polyacrylamide gels of the proteins from tunicamycin-treatedcells showed increased mobility and the absence of [³H]mannoseincorporation of the immunoglobulin heavy chains, consistentwith the lack of glycosylation. The native and non-glycosylatedantibodies were then tested for their ability to bind differentantigens. Despite the lack of glycosylation, both polyreactiveand monoreactive antibodies bound to antigens with little ifany loss of reactivity or specificity. It is concluded thatthe carbohydrate moieties do not contribute significantly topolyreactivity. antigen binding glycosylation polyreactive antibodies