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At least 343 records · Page 19Linked to original sources
PubMed1989

[Distribution of C3b receptors on polymorphonuclear leukocytes from normal healthy adults by scanning immunoelectron microscopy].

The distribution of C3b receptors on polymorphonuclear leukocytes cultured from normal healthy volunteers was examined by scanning immunoelectron microscopy with mouse anti-CR1 monoclonal antibody and 40 nm-goat anti-mouse IgG antibody. The distribution was categorized into three types; first, Au-particles clustered in numbers of several to ten or more on polymorphonuclear leukocytes (mean +/- S.D. = 59.5 +/- 36.8, n = 146), second, tens of the particles mostly on two or three ruffles of the cells (121.7 +/- 63.8, n = 45), and third, particles assembled at one part of the cells (74.1 +/- 47.4, n = 59). The distribution of Au-particles on all polymorphonuclear leukocytes showed a normal distribution curve (74.1 +/- 50.6, n = 250, p less than 0.02). These results show that polymorphonuclear leukocytes had some tendencies but no uniformity of distribution of C3b receptors on their plasma membrane when they were cultured.

Cells, Cultured↗
PubMed1985

Scanning immunoelectron microscopy of hairy cell leukemia.

Scanning electron microscopy has shown a typical cell surface morphology in hairy cell leukemia. Scanning immunoelectron microscope techniques, utilizing monoclonal antibodies and colloidal gold particles, have recently become available. Eight patients with hairy cell leukemia have been studied with a panel of monoclonal antibodies of which B1, BA1, OKM1, anti-TAC and LeuM5 were shown to be suitable for scanning immunoelectron microscopy and reactive with hairy cells. The combination of typical cell surface features with reactivity for the B1 and LeuM5 monoclonal antibodies permits accurate diagnosis of hairy cell leukemia, particularly when low percentages of hairy cells are present as in hypocellular or in interferon-treated patients.

Antibodies, Monoclonal↗
PubMed1985

Approach to the direct intramolecular localization of antigenic determinants in Androctonus australis hemocyanin with monoclonal antibodies by molecular immunoelectron microscopy.

Monoclonal antibodies (mAb) directed vs. subunits from hemocyanin (Hc) of the scorpion Androctonus australis were used in molecular immunoelectron microscopy (MIEM) to directly localize the epitopes within the subunits. Four types of mAb were used. First, mAb 6302, an IgG clone highly specific for subunit Aa 2, produced with native hemocyanin long strings composed of hemocyanin molecules in the side view and in the 45 degrees view. At lower concentration, "parachute" and "butterfly" structures composed of two Hc molecules and one monoclonal immunoglobin G (IgG) molecule were obtained. Fab fragments prepared from mAb 6302 bound exactly on the top and bottom edges of the molecule. The second type of mAb (6003), directed vs. subunit Aa 2, produced nice immunocomplexes with the free subunit but nothing with the native oligomer. It is suggested that due to steric hindrance or to conformational changes the epitope is not accessible in the native molecule. The third mAb belonged to the IgM class and apparently bound Hc in the Aa 2 area. However, because of the difficulty of separating the immunocomplexes from the residual mAb and the polymorphism of the IgM molecules, monoclonal IgM are no longer used for MIEM. The last type of mAb (5701) had a high affinity and a high specificity for subunit Aa 6. It produced two types of immunocomplexes with native Hc. The two types differed by a 180 degrees rotation around one of the Fab arms. These complexes, which support recent results of Wrigley et al. [Wrigley, N. G., Brown, E. B., & Skehel, J. J. (1983) J. Mol. Biol. 169, 771-774] and of Roux [Roux, K. H. (1983) Eur. J. Immunol. 14, 459-464], indicate that monoclonal IgG have a high degree of rotational flexibility around the Fab arm. Monoclonal antibody 5701 bound exactly at the corner of the molecule in the area where subunit Aa 6 is known to be located. The MIEM approach of the location of the epitope requires the model of the architecture and of the quaternary structure to be very precise. Thus, recent findings of Gaykema et al. [Gaykema, W. P. J., Hol, J. M., Vereijken, J. M., Soeter, N. M., Bak, H. J., & Beintema, J. J. (1984) Nature (London) 309, 23-29] and of Van Heel et al. [Van Heel, M., Keegstra, W., Schutter, W., & Van Bruggen, E. F. J. (1983) Life Chem. Rep., Suppl. Ser. 1, 69-73] led to a reexamination of previous models.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1985

Ultrastructural localization of dipicolinic acid in dormant spores of Bacillus subtilis by immunoelectron microscopy with colloidal gold particles.

The localization of dipicolinic acid in dormant spores of Bacillus subtilis was examined by an immunoelectron microscopy method with colloidal gold-immunoglobulin G complex. The colloidal gold particles were distributed mainly in the core regions of dormant spores and were not observed in those of germinated or autoclaved spores. This result clearly demonstrates that dipicolinic acid is localized in the cores of dormant spores.

Bacillus subtilis↗
PubMed1979

Scanning immunoelectron microscopy markers.

Ultrastructural analysis of the cell surface and of membrane components such as antigens or receptor sites by scanning immunoelectron microscopy (immuno-SEM) has been the subject of extensive investigation during the past few years. We review the various immunologic and cytochemical techniques applied to SEM, which have employed latex particles, viruses, bacteriophages, protein A, ferritin, gold granules, hemocyanin and peroxidase as markers, and the advantages and disadvantages of these techniques. From current data, it is clear that immuno-SEM has much to offer in determining the distribution of specific cell surface sites and in positive and unambiguous identification of cell types in heterogeneous cell populations.

Antigen-Antibody Reactions↗
PubMed1988

The use of colloidal gold immunoelectron microscopy to diagnose varicella-zoster virus (VZV) infections by rapid discrimination between VZV, HSV-1 and HSV-2.

Colloidal gold immunoelectron microscopy was used to diagnose rapidly 53 cases clinically suspected of varicella-zoster virus (VZV) infection and one special case selected from another study on typical herpes simplex virus (HSV) infections. The viruses were identified and subsequently typed within 2.5 h by a direct labelling test for VZV, and within 3.5 h by an indirect labelling test with monoclonal antibodies against HSV type 1 and type 2. The protein A purified IgG fraction of human anti-VZV immunoglobulins was adsorbed to colloidal gold particles, and the specificity of the gold-labelled antibodies was tested with several human and animal herpesviruses. Viral envelopes did not crossreact in the direct labelling test. However, an indirect labelling procedure revealed that a small fraction of the anti-VZV antibodies crossreacted with the cores of herpes simplex virus and pseudorabies virus (Aujeszky disease virus). Virus-infected cellular material taken from typical herpetic lesions was used directly without virus propagation for virus typing. All cases (N = 54) were analyzed without knowing the clinical description of the results of cytopathologic examination (Tzanck smear) and viral culture. Forty-four cases were identified as VZV; however, 5 of the supposed VZV infections were proved to be HSV infections. Although the viral culture of the one HSV case was HSV- and VZV-negative, colloidal gold labelling identified the case as VZV infection. In 16 cases virus immunoglobulin complexes were detected by using gold-tagged antibodies against human immunoglobulins. Immunoglobulins on the viral envelopes did not interfere with virus typing by immunogold labelling.

Antibodies, Viral↗
PubMed1982

Ribosomal protein S4 is an internal protein: Localization by immunoelectron microscopy on protein-deficient subribosomal particles.

The location of protein S4 in the small ribosomal subunit has been identified by immunoelectron microscopy. Although intact small subunits are not reactive with antibodies directed against protein S4, subribosomal particles reconstituted without proteins S5 and S12 are reactive. By using these "incomplete" subparticles, we have mapped the position of S4. It is located at a single site on the exterior (cytoplasmic) side of the subunit, at the partition that separates the one-third, or head, from two-thirds, or base, of the subunit. In this location, protein S4 is "beneath" proteins S5 and S12. All three proteins are members of a complex on, or near, the external surface of the small ribosomal subunit that plays an important role in regulation of translational fidelity.

Electrophoresis, Polyacrylamide Gel↗
PubMed1987

Visualization of the cytoplasmic surface of Torpedo postsynaptic membranes by freeze-etch and immunoelectron microscopy.

The synapse-specific Mr 43,000 protein (43K protein) and the acetylcholine receptor were visualized by freeze-etch immunoelectron microscopy in preparations of purified Torpedo postsynaptic membranes. Vesicles were immobilized on glass and then sheared open by sonication to expose the cytoplasmic surface. Membranes were labeled with monoclonal antibodies to the 43K protein or the acetylcholine receptor. The cytoplasmic surface was devoid of filamentous structure, and the 43K protein and the cytoplasmic projection of the acetylcholine receptor were associated with prominent surface particles. Acetylcholine receptor and 43K protein, in membrane surfaces in direct contact with glass coated with polyornithine, segregated into dense particle aggregates separated by smooth membrane patches, whereas those in contact with glass coated with Alcian Blue underwent little or no detectable rearrangement. After treatment of vesicles at alkaline pH to remove the 43K protein, the cytoplasmic surfaces were still covered by a dense array of particles that were more uniform in shape and appeared slightly shorter than those seen on unextracted membranes, but similar in height to the extracellular projection. Monoclonal antibodies to the acetylcholine receptor labeled these particles, while antibodies to 43K protein did not. We conclude that the 43K protein is in direct association with the receptor and that complexes of the receptor and 43K protein can undergo surface-induced lateral redistribution. In addition, the cytoplasmic projection of the acetylcholine receptor is sufficiently large to be readily detected by freeze-etch electron microscopy and is similar in height to the extracellular projection.

Animals↗
PubMed1984

Direct demonstration of multiple VH allotopes on rabbit Ig molecules: allotope characteristics and Fab arm rotational flexibility revealed by immunoelectron microscopy.

Immune complexes composed of rabbit IgG and Fab fragments of antibody specific for the VH framework allotypes were analyzed by molecular immunoelectron microscopy. In this manner, the number of allotypic epitopes ( allotopes ) and their approximate topological location could be determined. A monoclonal anti-allotype Fab was used to show that relatively fine details of allotope location and orientation are demonstrable with this technique. Each of the three VH allotypes (a1, a2 and a3) was found to express at least two spacially separated allotopes . The locations of the allotopes varied for each of the three allotypes. Some allotopes were observed near the VH-CH1 switch region while others were in close proximity to the complementarity-determining region or were at intermediate positions. In addition to the information concerning allotope topology, the variety of configurations resulting from the interaction of monoclonal antibody Fab with IgG suggest considerable rotational flexibility (twisting) of the Fab arm of IgG about its long axis.

Animals↗
PubMed1985

Morphology and immunoelectron microscopy of AIDS virus.

Lymphocytes infected with human T-lymphotropic virus type III/lymphadenopathy-associated virus were examined by thin-section and immunoelectron microscopy, with horseradish peroxidase as an electron-dense marker. There was no evidence of an intracytoplasmic virion precursor, but viral antigen accumulated at the plasma membrane. Particles formed at the plasma membrane, and most virus was found in extracellular spaces. Mature virus had an envelope with fuzzy surface projections surrounding a centric core and an electron-dense eccentric nucleoid.

Antibodies, Viral↗
PubMed1976

Chromatin structure visualization by immunoelectron microscopy.

Antibodies elicited in rabbits by chromatin and by purified histone H2B have been used to study the structure of chromatin by immunoelectron microscopy. Chromatin spread on grids reveals a structure of closely packed spherical particles with an average diameter of 104 A, arranged either in clusters or in linear arrays of beads, some of which have a supercoil-like arrangement. No DNA strings connecting the beads could be observed. Upon antibody binding, the diameter of the particles increases up to 300 A. This size is compatible with a model where one layer of gamma globulin molecules 110 A long encircles a sphere of chromatin 100 A in diameter. The presence of rabbit gamma globulins on the enlarged beads has been verified by the addition of ferritin-labeled goat anti-rabbit gamma globulins. Anti-chromatin sera which react with nonhistone proteins but not with free histones or DNA react with more than 95% of the beads; this suggests that most of the beads contain nonhistone proteins. Since the number of nonhistone proteins is large, it is improbable that each sphere contains a full complement of these proteins. We therefore suggest that the various chromatin spheres contain different types of nonhistone proteins. About 90% of the chromatin spheres reacted with antibodies to histone H2B, suggesting the most of the chromatin beads contain this type of histone.

Antigen-Antibody Reactions↗
PubMed1975

Localization of secretory IgA, secretory component, and alpha chain in the mammary gland of lactating rabbits by immunoelectron microscopy.

The different steps in the processing of secretory IgA (sIgA) of the mammary gland of lactating rabbits have been investigated with immunoelectron microscopy. The method employed consists of an indirect localization sequence, in which the immunologic reagents are allowed to diffuse into the fixed tissue prior to embedding and thin sectioning. In the first step, goat F(ab')2 fragments directed against sIgA, secretory component (SC), or alpha chain were used. To visualize the location of these first-step antibody fragments, a second Fab fragment directed against goat F(ab')2 antibody was prepared and coupled to a small heme octapeptide that possessed peroxidatic activity, the reaction product of which is visible by electron microscopy. Both the epithelial cell and the local plasmacytes of the mammary gland contain alpha chain, whereas SC is exclusively located in the epithelial cells. In the plasmacytes, both the cisternae of the rough endoplasmic reticulum and the saccules and vesicles of the Golgi complex contain alpha chain. In the epithelial cells, the elements of the Golgi complex and large apical vacuoles situated in the apical region of the cell. Based on these results, a model for processing of sIgA is proposed and discussed.

Animals↗
PubMed1986

Studies of tektin filaments from flagellar microtubules by immunoelectron microscopy.

Chemically resistant 2-3 nm filaments with a high alpha-helical content, isolated from sea-urchin sperm flagellar doublet microtubules, consist of proteins that have been named tektins. Polyclonal affinity-purified antibodies to tektins labelled sperm tails all along their lengths, as shown by indirect immunofluorescence microscopy, provided the specimens were not too well fixed. Results obtained for unfixed specimens studied by immunoelectron microscopy suggested the tektins are normally masked by tubulin. A monoclonal anti-tektin antibody labelled bare tektin filaments at longitudinal intervals of approximately 48 nm, which fits in well with the 96 nm longitudinal repeat of axonemes. We discuss a possible scheme for the regular interaction of tubulin monomers with an alpha-helical coiled coil.

Animals↗
PubMed1986

Macrophage type 3 complement receptors mediate serum-independent binding of Leishmania donovani. Detection of macrophage-derived complement on the parasite surface by immunoelectron microscopy.

In this study, direct visual evidence for local opsonization of L. donovani by macrophage (M phi)-derived complement components was obtained using immunoelectron microscopy. C3 deposition was detected on the surface of both promastigotes and amastigotes after 20 min serum-free incubation with murine resident peritoneal M phi (RPM), followed by fixation and incubation first with specific antibody directed against C3 and then with gold-labelled protein A. Gold deposition was not observed around either form of the parasite if the anti-C3 antibody was omitted. For promastigotes, the degree of C3 deposition under serum-free conditions was comparable with that observed in the presence of an exogenous (serum) source of C3, but did not result in the same severe damage to the parasite as did the latter. Addition of sodium salicyl hydroxamate, which prevents covalent binding of C3 to activator surfaces, abrogated promastigote binding. Hence, although the anti-C3 antibody did not distinguish between native C3 and its breakdown product iC3b, these data support our earlier conclusion that promastigote binding to the CR3 of murine RPM is complement dependent. For amastigotes, gold deposition and binding to murine RPM were not eliminated by sodium salicyl hydroxamate. The presence of normal mouse serum resulted in increased gold deposition, but did not mediate either enhanced binding to M phi or damage to the amastigote. These data suggest that a proportion of C3 binding to the amastigote surface may be via noncovalent linkages, and that the C3 bound may not be in the correct form to mediate binding to CR3.

Animals↗
PubMed1989

Prostaglandin I2 synthase in the lymphatic endothelium of rat liver as revealed by preembedding immunoelectron microscopy.

Localization of prostaglandin I2 synthase-like immunoreactivity (PGI2SI) in the lymphatic endothelium of rat liver was investigated by means of preembedding immunoelectron microscopy. Vesicular and multivesicular bodies with PGI2SI were closely apposed to Golgi complexes of the endothelial cells. Membrane-bounded PGI2SI structures including vesicular and dense bodies were also present beneath the endothelial plasmalemma on both luminal and abluminal sides. Other organelles within the endothelial cells did not exhibit PGI2SI. These findings are consistent with previous biochemical and physiological studies suggesting prostaglandin I2 is produced in the Golgi apparatus and released into luminal and abluminal spaces through membrane-bounded structures containing PGI2SI.

Animals↗
PubMed1989

Development of protein A-gold immunoelectron microscopy for detection of bovine coronavirus in calves: comparison with ELISA and direct immunofluorescence of nasal epithelial cells.

A protein A-colloidal gold immunoelectron microscopy (PAG-IEM) technique was developed for the detection of bovine coronavirus (BCV) in the feces and nasal secretions of infected calves. Feces or nasal swab fluids were incubated sequentially with hyperimmune bovine anti-bovine coronavirus serum and protein A-gold, negatively stained, applied to formvar-coated copper grids and viewed using an electron microscope. The PAG-IEM method specifically identified BCV particles and possible subviral particles in feces and nasal-swab fluids from infected calves. The PAG-IEM method did not label other enveloped enteric viruses or morphologically similar fringed particles commonly found in feces. Detection of BCV using PAG-IEM was compared with ELISA and direct immunofluorescence (IF) of nasal epithelial cells by monitoring fecal and respiratory tract shedding of BCV from two experimentally infected and two naturally infected calves from birth to 3 weeks of age. PAG-IEM and ELISA detected shedding of BCV in fecal (4/4 animals) and nasal (3/4 animals) samples for an average of 5.25 days each. The observed agreement of BCV detection by PAG-IEM and ELISA was 85%. PAG-IEM may be a more sensitive immunoassay for the detection of BCV in diagnostic specimens from infected neonatal calves than ELISA. BCV infection of nasal epithelial cells was detected by immunofluorescence in 4/4 calves, persisted for the duration of the study in 2/4 calves and was sporadic in the other two animals. The observed agreement of BCV detection by PAG-IEM and IF was 57%.

Animals↗
PubMed1973

Demonstration of a cell-surface antigen associated with murine sarcoma virus by immunoelectron microscopy.

Cells transformed by murine sarcoma virus have been examined for the presence of a new virus-associated cell-surface antigen by immunoelectron microscopy. A common antigen has been detected on the surface of nonproductively transformed cells that were induced by two different strains of murine sarcoma virus, Kirsten and Moloney. This antigen shows crossreaction with cell lines transformed by murine sarcoma virus that were produced in two different mammalian species, rats and mice. Further, this antigen is distinct from previously described antigens on the surfaces of cells infected by murine leukemia virus, on the viral envelope, and on the surfaces of spontaneously transformed cell lines or cell lines transformed by x-irradiation.

Animals↗