Colloidal gold probes for parasite antigens.
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Journal Article↗
Biomedical subjects
J E Beesley
Publications and source records attributed to J E Beesley.
Purification, characterization and immunological properties of the capsular polysaccharide of Pasteurella haemolytica serotype T15: its identity with the K62 (K2ab) capsular polysaccharide of Escherichia coli and the capsular polysaccharide of Neisseria meningitidis serogroup H.
Capsular polysaccharide from two strains of Pasteurella haemolytica serotype T15 was purified and characterized by chemical analysis and NMR spectroscopy. The polymer, a teichoic acid, proved to be very similar in structure to the capsular polysaccharide of P. haemolytica serotype T4 and identical to the previously described K62 (K2ab) capsular polysaccharide of Escherichia coli, and the capsular polysaccharide of Neisseria meningitidis serotype H, i.e. ----(2-glycerol-3)----(phosphate)----(4-alpha-D-galactopyranose -1)---- with partial O-acetylation on the galactose residues. Electron microscopy with Protein A-gold labelled antisera showed that the polysaccharide was peripherally located on the surface of all three organisms. Chemical removal of O-acetyl groups from the polysaccharide yielded a structure identical to that previously described for E. coli K2 (K2a). Both O-acetylated and de-O-acetylated P. haemolytica T15 polymers, when absorbed on to sheep erythrocytes in passive haemagglutination assays, yielded identical antibody titres with sera raised against P. haemolytica T15, E. coli K2 or N. meningitidis H whole cells. De-O-acetylation of the Pasteurella polysaccharide influenced its precipitability with immune sera, but this could not be related to the absence of O-acetyl groups because the non-acetylated E. coli K2 polymer readily precipitated with a line of 'identity' with the acetylated P. haemolytica T15 polymer.
Escherichia coli↗
The accumulation of an aryloxylkylamidine (501C) and 5-hydroxytryptamine in human polymorphonuclear leucocytes: a quantitative electron microscopy study.
The uptake of anilino-N-2-m-chlorophenoxypropylacetamidine (501C) and 5-hydroxytryptamine (5-HT), in human polymorphonuclear leucocytes (PMN) was investigated by quantitative ultrastructural autoradiography. The major concentration of both drugs was in the cytoplasmic granules, 71.5 +/- 6.8% for [3H] 501C, and 68.75 +/- 6.1% for [3H]5-HT. Lesser quantities of both drugs were associated with the nucleus; 18.1 +/- 4.8% for [3H] 501C and 23.0 +/- 4.2% for [3H] 5-HT. Only small amounts of activity were recorded at other sites. The data suggests that there may be common binding sites for 501C and 5-HT in PMN. Furthermore the concentration of 501C in PMN granules may also account for the damaging effect of high concentrations of the drug on PMN.
Amidines↗
Localisation of Ca and HMFG2 antigens in breast tissue by immunoperoxidase, immunofluorescence, and immunoelectron microscopy.
The reactivities of Ca1 and HMFG2 monoclonal antibodies were compared on paraffin wax embedded breast tissues using indirect immunoperoxidase. The expression of Ca antigen, like HMFG2, is not exclusive to malignancy: Ca was present in 41/53 (77%) and HMFG2 in 42/53 (79.2%) non-malignant conditions and both were present in 33/35 (94%) carcinomas. Similar results were obtained when cryostat sections were used. Both antigens showed striking similarities in their topographical distributions, although quantitative differences were seen. Their cellular and sub-cellular localisations were investigated by double labelling immunofluorescence and immunogold electron microscopy, which showed that the expression of Ca and HMFG2 antigens was closely associated on cell membranes but that the epitopes were distinct.
Adenofibroma↗
Bordetella adenylate cyclase: a genus specific protective antigen and virulence factor.
Most of the adenylate cyclase (AC) present in Bordetella species is localized in the outer membrane, partly exposed to the cell surface. An isolation procedure to obtain the cell-bound enzyme was applied to Bordetella bronchiseptica, Bordetella pertussis and Bordetella parapertussis. Passive transfer of B. bronchiseptica anti-AC antibody, either in the form of monoclonal antibody or antibody transferred to offspring from female mice previously immunized with immunopurified B. bronchiseptica AC, prevented both death and the development of "atrophic rhinitis" in mice infected by an aerosol of virulent B. bronchiseptica. Immunopurified AC from both B. bronchiseptica and B. pertussis protected mice against intracerebral challenge with strain 18323, although five to six times more protein of B. bronchiseptica was necessary to produce the same level of protection as with B. pertussis-derived protein. The AC of Bordetella species differ in molecular weight and, despite sharing at least two common non-overlapping epitopes (BB05, BB07), antigenic differences are likely: sera from piglets artificially infected with virulent B. bronchiseptica developed high ELISA titers against AC from B. bronchiseptica but low titers against B. pertussis-derived antigen. Strains of B. bronchiseptica which did not induce "atrophic rhinitis" in pigs were found to be AC-less variants. When tested in our laboratory model, such a strain was also unable to induce "atrophic rhinitis" in mice. We conclude that AC is a mandatory component of any future subcellular vaccine against Bordetella species.
Adenylyl Cyclases↗
Virus diagnosis: a novel use for the protein A-gold probe.
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Animals↗
The use of ultrathin cryosections for localisation of influenza virus antigens in infected vero cell cultures.
The localisation of influenza virus antigens in infected Vero cell monolayer cultures by post embedding immunoelectron microscopy requires both good resolution and the retention of antigenicity in the tissue sections. Ultrathin cryosections are superior to ultrathin resin sections for this purpose. The colloidal gold probe was used in conjunction with specific antibody preparations to localise three viral proteins. Antibody raised against haemagglutinin glycoproteins labelled the host cell membrane and the virus fringe without contamination of the host cell nucleus, whereas antibody raised against viral nuclear protein labelled throughout the host cell cytoplasm and nucleus. Matrix protein was localised within the nucleus and was associated with the host cell membrane of the infected cell. The appearance of all these proteins was maximal 24 h post infection.
Animals↗
An evaluation of the conditions necessary for optimal protein A-gold labelling of capsular antigen in ultrathin methacrylate sections of the bacterium Pasteurella haemolytica.
The protein A-gold (PAG) probe is a particulate immunocytochemical probe that is eminently suitable for quantification. In order to obtain critical results from the technique, a specific and reproducible probe is needed. To this end, the concentration of probe, the variation of labelling on different sections within a single grid, the effect of washing procedures, the variation of labelling with time and temperature and the effect of different storage conditions on the probe have been investigated using PAG labelling of capsular antigen on ultrathin methacrylate sections of the bacterium Pasteurella haemolytica. The results indicate that in this antigen-antibody system, and using a 20 nm probe, optimal results are achieved with 2 X 10(12) particles/ml, a labelling time of 60 min at room temperature and the PAG probe, which will have been stored at 4 degrees C, should be between 1- and 5-weeks-old. The efficiency of the probe is tested by evaluating different primary antibody concentrations, by evaluating cross reactions of the primary antibody and by evaluating the relative amounts of antibody against internal components of the bacterium present in different antisera.
Antigens, Bacterial↗
Immunocytochemical labelling of Bacteroides nodosus pili using an immunogold technique.
An immunogold labelling technique was used to label the pili of the bacterium Bacteroides nodosus. The labelling was distinct and highly specific, and individual pili could be recognised beneath the gold probe. The labelling of somatic antigens could be distinguished from that of pilus antigens. Furthermore, labelling of fragments of cytoplasm released by cell lysis and trapped in the pili could be distinguished from pilus labelling. An antiserum that had been raised against strain 80200 (serotype N) labelled pili of strain 215 (serotype B). Double labelling experiments with this antiserum and the antiserum against strain 215 (serotype B) showed that both antisera label the same pili bundles. The ease of detection of the immunocytochemical reaction should enable this technique to be used as a routine screen for pilus antigens. It also possesses the potential for much wider applications for immunolabelling other antigens, such as viruses, that can be obtained in suspension.
Bacteroides↗
Purification, characterization and immunological properties of the serotype-specific capsular polysaccharide of Pasteurella haemolytica (serotype A1) organisms.
The serotype-specific capsular polysaccharide from two strains of Pasteurella haemolytica serotype A1 organisms was purified and characterized by chemical analysis and NMR spectroscopy. The polymer has the structure----3)-O-(2-acetamido-2-deoxy-4-O-acetyl-beta-D-mannopyranos yluronic acid)-(1----4)-O-(2-acetamido-2-deoxy-beta-D-mannopyranose)-(1----. The polysaccharide was immunogenic (able to evoke production of antibodies) for sheep but not for rabbits. Immuno electron-microscopy studies using the Protein A-gold technique showed the polysaccharide to be peripherally located on the bacterial surface. Reduction, oxidation and de-O-acetylation of the polymer did not appear to alter its immunological precipitability with specific antiserum, but all three treatments destroyed its ability to adhere to sheep erythrocytes at neutral pH. De-N-acetylation of the polymer destroyed both immunological precipitability and erythrocyte adherence.
Amino Acids↗
Trends in the localization of bacterial antigens by immunoelectron microscopy.
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Antigens, Bacterial↗
The sporulation of Eimeria tenella as revealed by a novel preparative method.
A simple technique for obtaining ultra-thin sections of coccidian oocysts is reported. Oocyst walls, the main barriers to fixatives, dehydrating agents and embedding media are ruptured by brief immersion of the organism in liquid nitrogen. Adequate penetration of chemicals is thus assured but with minimal damage to the internal tissue. The fine structure of sporulation of oocysts of Eimeria tenella was studied using this process. Each oocyst produced four sporoblasts. These developed into sporocysts and each produced two sporozoites.
Animals↗
A comparison of immunoferritin, immuno-enzyme and gold-labelled protein A methods for the localization of capsular antigen on frozen thin sections of the bacterium, Pasteurella haemolytica.
The staphylococcal protein A-gold method was found to be superior to the enzyme-or ferritin-linked antibody techniques for locating capsular antigens on cryosections of Pasteurella haemolytica, and its sensitivity was similar to the enzyme-linked antibody method. The sensitivity of conventionally fixed and embedded material and cryosections of heavily fixed, lightly fixed and unfixed material were shown to be similar under routine laboratory conditions.
Antigens, Bacterial↗
The ultrastructure of feline infectious peritonitis virus in feline embryonic lung cells.
The ultrastructure of feline infectious peritonitis virus in cultured feline embryonic lung cells is reported. Feline embryonic lung cells were infected with feline infectious peritonitis virus and studied by transmission electron microscopy. The virus was not apparent in the cultured cells until 24 h after infection when it occurred in the endoplasmic reticulum, perinuclear space, Golgi apparatus, free in the cytoplasm, in large vacuoles in the cytoplasm and outside the cell membrane. The virus possessed typical coronavirus morphology and was produced by budding into the endoplasmic reticulum. There was no evidence to indicate that this virus budded through the cell membrane. Multinucleate giant cells were formed by infection of the cultured cells with the virus. The host cells were destroyed by the virus and phagocytosed by apparently healthy cells.
Animals↗
In vitro culture of feline infectious peritonitis virus.
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Animals↗
Ultrastructural localization of characterized antigens of Brucella abortus and distribution among different biotypes.
The distribution of characterized antigens in extracts of representative strains of Brucella abortus biotypes 1 to 9 was investigated by hemagglutination and precipitation reactivity. An antigen designated X was present in high concentrations in sodium dodecyl sulfate extracts of all smooth strains examined, but in low concentrations in the rough strain 45/20 extract. Antigen beta was demonstrable in 8 out of 12 smooth strains of B. abortus, but antigen gamma was detected in only 3 of these strains. Vaccine strain 19 extract contained beta antigen but no detectable gamma antigen, whereas both these antigens were present in rough strain 45/20 at slightly lower concentrations that in strain 544/W extract. Immunoenzyme histochemical studies performed with an electron microscope showed that antigens beta, gamma, and X were all situated in the outer membrane envelope of the cell wall of B. abortus 544/W.
Animals↗
Regulation of macrophage lysosomal enzyme secretion: role of arachidonate metabolites, divalent cations and cyclic AMP.
We have investigated the role in macrophage lysosomal enzyme release of arachidonate metabolites, extracellular divalent cations and cyclic AMP (cAMP) which modulate secretion in other cell types. Lysosomal enzyme secretion induced by zymosan was accompanied by release of malondialdehyde (MDA), which is derived from arachidonic acid via prostaglandin synthase. Blockade of MDA formation, by aspirin or indomethacin, was associated with only a small inhibitory effect on lysosomal enzyme release by zymosan: arachidonate metabolites thus play only a minor role in mediating macrophage lysosomal enzyme release. Zymosan-induced secretion of lysosomal enzymes from macrophages did not require extracellular magnesium or calcium although release was enhanced by magnesium and inhibited by calcium. These effects may be related to an influence of the ions on phagocytosis. Elevation of intracellular divalent cation concentrations, by ionophore A23187, induced release of lysosomal enzymes but this was a result of cell lysis. Adenylate cyclase stimulants and dibutyryl cAMP produced slight inhibition of zymosan-induced lysosomal enzyme release. Aminophylline and papaverine caused more marked inhibition but their effects may be due to actions independent of phosphodiesterase inhibition. Our data indicate that arachidonate metabolites and cAMP do not play a major role in regulating zymosan-induced enzyme release from macrophages. Extracellular calcium and magnesium may modulate secretion but the role of intracellular divalent cations remains to be established. We conclude that macrophage lysosomal enzyme secretion is controlled by regulatory mechanisms different from those which control similar degranulation processes in other cell types.
Animals↗
Granulocyte adhesion to endothelium in culture.
We previously described a model system for studying the adherence of granulocytes to cultured endothelium, and have now investigated the effects of other blood components on granulocyte adhesion in this system. Red cells enhanced adhesion, whereas blood platelets decreased adhesion, and further experiments suggested that endothelial cells secrete a proadhesive factor, particularly if incubated with plasma. We have also investigated the effects of several drugs, and attempted to localize their sites of action. Flavonoid drugs increased adhesion by an effect at the endothelial cell surface, whereas agents that increase cyclic AMP levels, including several prostaglandins, stimulated adhesion mainly by their effects on granulocytes. The effects of some agents on granulocyte adhesion to endothelium were not paralleled by their effects on adhesion to serum-coated glass. We conclude that granulocyte-endothelial interaction is a complex process, with each cell type responding to the other or to factors produced by it, and that data derived solely from studies of granulocyte adhesion to inert substrata will not always reflect granulocyte-endothelial interaction in vivo.
Animals↗