Monoclonal antibodies against species-specific antigens in the chick central nervous system: putative application as transplantation markers in the chick-quail chimera

With the recent progress in transplantation of neuronal tissues, cellular markers are needed to distinguish the grafted cells from the host. To generate monoclonal antibodies (MAb) recognizing species-specific antigens in the chick nervous system, we immunized mice with chick optic nerves and obtained 2 MAb which bind to chick but not to quail neural tissues. MAb-39B11 recognizes the cell surface antigen on the nerve fibers. MAb-37F5 recognizes the cytoplasmic components in several cell types, including ependymal cells and some large neurons. The utility of these MAb as markers for chick cells in the chick-quail chimeric brain and their advantages over conventional markers are discussed.     

Changes in cell surface glycoproteins during Dictyostelium development analysed using monoclonal antibodies

We have produced a series of monoclonal antibodies that recognize carbohydrate epitopes on cell surface glycoproteins of developing amoebae of Dictyostelium discoideum. The antibodies were found to have differential specificity for amoebae at different stages of development and were classified into types A to E on the basis of their temporal pattern of reactivity with the developing amoebal cell surface. Evidence from Western Blots and digestion of the glycoproteins with alkaline phosphatase were consistent with previous reports that the cell surface glycoproteins are extensively processed during development, leading at 16 h of development to the exposure of a highly antigenic core recognized by antibodies in group E. The nature of this core structure is indicated by the finding that antibodies in group E were found also to bind with high avidity to the plant glycoprotein horse radish peroxidase.     

Subspecies- and species-specific antigens of Leishmania mexicana characterized by monoclonal antibodies

Monoclonal antibodies have been produced that are specific for the reference stocks of Leishmania mexicana species and subspecies L. mexicana mexicana(L11, M379), L. mexicana amazonensis (WR303, H6, LV72), and L. mexicana pifanoi (L20). The specificities of these antibodies were confirmed by analyses employing an indirect radioimmune binding assay and 107 stocks of New World Leishmania. The molecules associated with these species- and subspecies-specific determinants have been characterized by Western blot analysis and consist of mainly low m.w. (11,000 to 50,000) membrane-associated components.     

Leishmania tropica: characterization of a lipophosphoglycan-like antigen recognized by species-specific monoclonal antibodies.

Species-specific monoclonal antibodies to Leishmania tropica, T11 and T13-15, recognize membranal and secreted antigens. The membrane form of the antigen migrates on sodium dodecyl sulfate-polyacrylamide gels with a diffuse molecular weight from 15 to 50 kDa and can be labeled with palmitic acid, myoinositol, galactose, glucosamine, and inorganic phosphate. Both phosphate and sugar-labeled material were isolated from metabolically labeled promastigotes by affinity chromatography on antibodies coupled to Sepharose 4B. No binding to Ricinus communis agglutinin was observed. This material behaves like lipophosphoglycans from other Leishmania but contains unique species-specific epitopes. It is susceptible to cleavage by phospholipase C and after digestion no longer partitions into the detergent phase following a Triton X-114 extraction. All four monoclonal antibodies appear to recognize a carbohydrate epitope on the lipophosphoglycan since periodate treatment of this material bound to nitrocellulose essentially eliminated antibody binding. In addition, T15 binding could be blocked by 5 mM mannose-6-PO4 and fructose-1- or 6-PO4, but not by mannose, glucose, fructose, or the additional PO4 derivatives examined. The antibodies recognize a similar but not identical epitope, as demonstrated by a competitive radioimmunoassay using 125I-labeled T11, T13, and T15. Expression of surface antigen is elevated during the promastigote stationary phase.     

Reactivity of monoclonal antibodies to species-specific antigens of Entamoeba histolytica.

Twenty monoclonal antibodies were produced against trophozoites of Entamoeba histolytica strains HK-9 and HM-1: IMSS. When reactivity to various enteric protozoa was examined by an indirect fluorescence antibody test, 15 of the monoclonal antibodies were strongly reactive with E. histolytica trophozoites. Species-specific antigens recognized by these monoclonal antibodies were located on the plasma membrane, nucleus, cytoplasm, and cytoskeletal structures of the trophozoites. Two of the remaining five monoclonals reacted strongly with trophozoites of the E. histolytica-like Laredo strain. The determinant antigen was located in the cytoplasm. The three remaining monoclonal antibodies were found to recognize cross-reactive antigens between E. histolytica and E. histolytica-like Laredo, E. hartmanni, E. coli, Dientamoeba fragilis, Giardia lamblia, and Trichomonas hominis. These three antibodies were also reactive with T. vaginalis and mammalian cells such as HeLa cells. Thus, the combined use of monoclonal antibodies seems capable of distinguishing E. histolytica and/or E. histolytica-like Laredo from other enteric protozoa.     

Immunoanatomic dissection of the human urinary tract by monoclonal antibodies

The immunoanatomy of the human kidney and urinary tract has been analyzed by a panel of mouse anti-human monoclonal antibodies that define specific domains and structures. The differentiation antigens detected by these monoclonal antibodies represent a series of glycoproteins characteristic of different cell types. They differ from the blood group antigens and appear to be distinct from other antigens previously described within the kidney or urinary tract. The antigens recognized by these monoclonal antibodies represent an immunohistologic dissection of the human nephron. These antibodies have a broad range of potential applications in studying embryogenesis and pathogenesis of nonneoplastic and neoplastic diseases of the human kidney and urothelium.     

Detection of fos protein during osteogenesis by monoclonal antibodies.

We have generated monoclonal antibodies by using a synthetic peptide corresponding to amino acid positions 4 to 17 of the human fos protein. The antibodies detected both v- and c-fos proteins by immunoprecipitation, immunoblotting, and indirect immunofluorescence. The monoclonal antibodies not only identified the fos protein complex with the cellular 39-kilodalton protein, but also recognized the modified forms of the mouse, rat, and human fos proteins. In day-17 rat embryos, nuclear-staining fos protein could be identified in the cartilage by immunohistochemical staining.     

Differentiation between thermophilic Campylobacter species by species-specific antibodies.

The four species of thermophilic campylobacters, Campylobacter jejuni, C. coli, C. upsaliensis and C. lari, are difficult to distinguish from each other because of their lack of reactivity in many conventional biochemical and physiological tests. Those tests which do discriminate sometimes give discordant results. Species-specific antibody preparations (APs), capable of discriminating between the thermophilic campylobacter species by dot-ELISA, were raised by inoculation of mice with partially purified membrane protein. The APs produced were absorbed with cells of cross-reactive species and tested by dot-ELISA against reference and natural strains, the identities of which were confirmed by DNA/DNA hybridization. The results showed that such APs could be useful as an alternative to DNA/DNA hybridization for rapid species identification, for example in epidemiological surveys. Western blotting experiments with the APs showed that the specificity of the antibodies was not due to a single antigen.     

Domain-and species-specific monoclonal antibodies recognize the Von Willebrand Factor-C domain of CCN5

The CCN family of proteins typically consists of four distinct peptide domains: an insulin-like growth factor binding protein-type (IGFBP) domain, a Von Willebrand Factor C (VWC) domain, a thrombospondin type 1 repeat (TSP1) domain, and a carboxy-terminal (CT) domain. The six family members participate in many processes, including proliferation, motility, cell-matrix signaling, angiogenesis, and wound healing. Accumulating evidence suggests that truncated and alternatively spliced isoforms are responsible for the diverse functions of CCN proteins in both normal and pathophysiologic states. Analysis of the properties and functions of individual CCN domains further corroborates this idea. CCN5 is unique among the CCN family members because it lacks the CT-domain. To dissect the domain functions of CCN5, we are developing domain-specific mouse monoclonal antibodies. Monoclonal antibodies have the advantages of great specificity, reproducibility, and ease of long-term storage and production. In this communication, we injected mixtures of GST-fused rat CCN5 domains into mice to generate monoclonal antibodies. To identify the domains recognized by the antibodies, we constructed serial expression plasmids that express dual-tagged rat CCN5 domains. All of the monoclonal antibodies generated to date recognize the VWC domain, indicating it is the most highly immunogenic of the CCN5 domains. We characterized one particular clone, 22H10, and found that it recognizes mouse and rat CCN5, but not human recombinant CCN5. Purified 22H10 was successfully applied in Western Blot analysis, immunofluorescence of cultured cells and tissues, and immunoprecipitation, indicating that it will be a useful tool for domain analysis and studies of mouse-human tumor models.     

Mechanism of chimera formation during the Multiple Displacement Amplification reaction

Background  

Multiple Displacement Amplification (MDA) is a method used for amplifying limiting DNA sources. The high molecular weight amplified DNA is ideal for DNA library construction. While this has enabled genomic sequencing from one or a few cells of unculturable microorganisms, the process is complicated by the tendency of MDA to generate chimeric DNA rearrangements in the amplified DNA. Determining the source of the DNA rearrangements would be an important step towards reducing or eliminating them.     

Extracellular amastigote-like forms of Leishmania panamensis and L. braziliensis. II. Stage- and species-specific monoclonal antibodies

Immunochemical evidence, employing monoclonal antibodies, shows that the forms of L. braziliensis complex axenically grown at elevated temperature are amastigote-like. The monoclonal antibodies were raised against membrane proteins of amastigote-like forms, strains of both L. panamensis (WR442) and L. braziliensis (M5052), which were grown axenically. The specificities of these antibodies were examined by indirect radioimmune binding assay, indirect immunofluorescent assay and Western blot analyses. Two distinct groups of monoclonal antibodies were obtained and their specificities were consistent with the 3 methods used. Four antibodies are specific for the species L. panamensis and react with both developmental stages. Six antibodies specifically recognize amastigote-like forms grown at elevated temperature and intracellular amastigotes of both L. panamensis (WR442) and L. braziliensis (M5052). These monoclonal antibodies do not bind to promastigotes of these species, nor to promastigotes of any other species of Leishmania. Therefore these antibodies are specific for amastigotes of L. panamensis (WR442) and L. braziliensis (M5052), and suggest that immunochemically both amastigote forms (culture and macrophage) are developmentally very close, if not identical. The molecules associated with the amastigote-specific antigenic determinants consist of a Mr 12-kD component and a heterogeneous component (Mr from 50 kD to greater than 200 kD); these molecules appear to be identical for both amastigote-like forms and amastigotes isolated from macrophages.     

Involvement of the alpha chain in fibrin clot formation. Effect of monoclonal antibodies.

Murine monoclonal antibodies 9C3, 7B1, and 9E9 have been obtained using native human fibrinogen as the antigen. The antibodies reacted with the epitopes in the COOH-terminal domain of the A alpha chain. Fragmentation of the A alpha chain with plasmin, and, as in the case of the 9E9 epitope, with V8 protease, followed by isolation of the smallest reacting peptides, allowed the localization of the epitopes for 9C3, 7B1, and 9E9 to the amino acid sequences of alpha 240-268, alpha 425-440, and alpha 541-574, respectively. All three monoclonal antibodies strongly inhibited the rate of fibrin polymer assembly from monomers, both in the purified system and in the human plasma. The mechanism of this strong inhibition implied a rapid formation of fibrin protofibrils, followed by capping with IgG molecules of protofibrils containing approximately ten monomers. These observations demonstrated that certain regions in the COOH terminus of the alpha chain may play an important role in the assembly of a fibrin clot, presumably being involved in lateral aggregation of protofibrils.     

T-cell depletion by monoclonal antibodies does not prevent granuloma formation in mice

Effects of T-cell depletion on the formation of organized granulomas in mouse skin were investigated. Monoclonal antibodies (MoAbs) to L3T4 and Lyt-2.2 were injected into euthymic BALB/c mice in order to deplete both T-helper and T-suppressor cell populations. Four days after injection, hepatic schistosome egg granulomas were transplanted into the skin. Injection of MoAbs to L3T4 and Lyt-2.2 was repeated in the recipient mice 6 days later. At the time of transplantation, flow cytometric analysis showed that the number of T cells which were positively stained with surface markers decreased on average by 68% in both regional lymph nodes and spleen. The mitogen response of spleen cells was also markedly reduced. Despite their immunosuppressed condition, development of organized granulomas was observed in the skin of recipient mice by light and electron microscopy 3 weeks after transplantation. The findings indicate that T-cell function may not be essential for initiation of organized granuloma formation.     

Expression of epithelial antigens in primary cultures of normal human breast analysed with monoclonal antibodies

Primary cultures of normal human breast were stained with monoclonal antibodies to see if antigens characteristic of luminal epithelial cells are retained in culture. Three monoclonal antibodies were used, LICR-LON-M8, LICR-LON-M18, and LICR-LON-M24, all specific for the cell surface of luminal epithelial as opposed to myoepithelial or stromal cells in the breast, and each staining a different subset of the epithelial cells in the intact tissue. Cultures were prepared from reduction mammoplasty samples by digestion with collagenase. The surface layer of cells was stained by immunofluorescence without fixation. (Cells underneath the surface layer were not accessible to this mode of staining). The antibodies stained patches of cells resembling flattened epithelium. These patches of cells cannot be distinguished by phase contrast microscopy without reference to the staining, in fact the boundaries of the cells are not usually resolved by phase contrast microscopy. Electron microscopy of sections through these cells show they are very flattened. They lie on top of the polygonal and elongated cells that dominate the phase contrast image. Two of the antibodies, M8 and M24, stain subsets of these epithelial-like cells at all stages of culture. The third antibody, M18, stains such cells initially, but after the first few days staining is predominantly found on the polygonal and elongated cells, then this also gradually disappears. It is possible that the cells stained by antibody M18 are converting from the epithelial-like morphology to the cuboidal and elongated morphology. Many cells are not stained by any of the antibodies, so appear either to by myoepithelial in origin or to have lost their luminal epithelial surface antigens at an early stage. This analysis draws attention to the variety of cell types in these cultures and the limitations of phase contrast microscopy as a means of analysing them.     

Expression of epithelial antigens in primary cultures of normal human breast analysed with monoclonal antibodies

Abstract. Primary cultures of normal human breast were stained with monoclonal antibodies to see if antigens characteristic of luminal epithelial cells are retained in culture. Three monoclonal antibodies were used, LICR-LON-M8, LICR-LON-M18, and LICR-LON-M24, all specific for the cell surface of luminal epithelial as opposed to myoepithelial or stromal cells in the breast, and each staining a different subset of the epithelial cells in the intact tissue. Cultures were prepared from reduction mammoplasty samples by digestion with collagenase. The surface layer of cells was stained by immunofluorescence without fixation. (Cells underneath the surface layer were not accessible to this mode of staining). The antibodies stained patches of cells resembling flattened epithelium. These patches of cells cannot be distinguished by phase contrast microscopy without reference to the staining, in fact the boundaries of the cells are not usually resolved by phase contrast microscopy. Electron microscopy of sections through these cells show they are very flattened. They lie on top of the polygonal and elongated cells that dominate the phase contrast image. Two of the antibodies, M8 and M24, stain subsets of these epithelial-like cells at all stages of culture. The third antibody, Ml8, stains such cells initially, but after the first few days staining is predominantly found on the polygonal and elongated cells, then this also gradually disappears. It is possible that the cells stained by antibody Ml8 are converting from the epithelial-like morphology to the cuboidal and elongated morphology. Many cells are not stained by any of the antibodies, so appear either to by myoepithelial in origin or to have lost their luminal epithelial surface antigens at an early stage. This analysis draws attention to the variety of cell types in these cultures and the limitations of phase contrast microscopy as a means of analysing them.     

Activation of beta-galactosidase by monoclonal antibodies.

Four monoclonal cell lines secreting antibodies that activate the beta-galactosidase protein from lac-aba strains of Escherichia coli have been isolated. One of the antibodies, BG 79, inhibits the normal beta-galactosidase from E. coli in addition to its activation of the protein from mutants. Moreover, when in combination with any of the other activating antibodies, BG 79 exhibits synergistic activation of the beta-galactosidase protein, and the synergistically activated enzyme is stimulated by methanol, although most of the proteins activated by single antibodies are inhibited by methanol. The equilibrium of binding of BG 79 to the beta-galactosidase protein is not affected by the presence of a second antibody, and the half-time for activation by BG 79 is only slightly, though significantly, increased by preincubation of the protein with the second antibody. Our results imply that activation of beta-galactosidase proteins is not a simple correction of a conformational defect, and that many distinct active conformations are available to the enzyme.     

Conformational changes induced by domain assembly within the beta 2 subunit of Escherichia coli tryptophan synthase analysed with monoclonal antibodies

The effects of domain assembly on the conformation of the F1 (N-terminal) and F2 (C-terminal) domains of the beta 2 subunit of Escherichia coli tryptophan synthase (EC 4.2.1.20) were analysed using six monoclonal antibodies which recognize six different epitopes of the native beta 2 subunit (five carried by the F1 domain and one carried by the F2 domain). For this purpose, the affinity constant of each monoclonal antibody for the isolated domains F1 or F2, the associated domains in the trypsin-nicked apo-beta 2 and in the native apo-beta 2 subunits were determined, both with the intact immunoglobulin and the Fab fragment. It was found that the association of the F1 and F2 domains within beta 2 is accompanied by structural changes of the two domains, as detected by variations of their affinity constants for the monoclonal antibodies.     

Hordein variation in the genus Hordeum as recognized by monoclonal antibodies.

The composition of the major storage protein, hordein, in wild barley species has been studied by using gel electrophoresis, Coomassie staining, and immunoblot assays. We have shown earlier that it is possible to obtain cross-reaction outside the cultivated barley, with monoclonal antibodies raised against hordeins from the barley cultivar Bomi. These antibodies have now been used to investigate the hordein composition in all species of the Hordeum genus. The results showed that polypeptides similar to the two major hordein groups of cultivated barley, the B- and C-hordeins, are produced in all wild Hordeum species, and that there are both similarities and differences between the two hordein groups. The similarities indicate a common evolutionary origin, while the distinction between B- and C-hordeins in the entire genus clearly shows that the divergence of their coding genes preceded the divergence of the Hordeum species. The presence of the same antigenic site in two different species indicates that they are evolutionarily related. Among the wild species, two rarely occurring sites were exclusively found in H. vulgare ssp. spontaneum and H. bulbosum, which confirms that they are the cultivated barley's closest relatives. Some of the antibodies also gave an extensive reaction pattern with H. murinum, which suggests a fairly close relationship to H. vulgare, though not as close as between H. vulgare and H. bulbosum.     

A novel method to bursectomize avian embryos and obtain quail----chick bursal chimeras. I. Immunocytochemical analysis of such chimeras by using species-specific monoclonal antibodies

Chick embryos were bursectomized at 5 days of incubation according to a novel surgical technique described in this article. This method yields birds that are able to hatch and are devoid of the physiologic deficiencies resulting from the previously used method, which involved resection of the cloacal and posterior embryonic region. The bursectomized embryos were grafted in situ with a quail bursa of the same age, which thereafter became chimeric through chick host hemopoietic cell invasion. By means of species-specific antibodies, the chimeric condition revealed 1) that the bursal epithelium expresses a unique antigenic determinant (MB1 determinant), until now considered to be an exclusive feature of blood vessel endothelium and hemopoietic cells, and 2) that this determinant appears in bursal epithelium at the time and site of hemopoietic cell invasion. The other point arising from this work concerns the apparent constitutive Ia expression by perifollicular blood capillary endothelial cells in normal and chimeric bursas.