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1.
Immunoglobulins M and G specific for meales, herpes simplex, and rubella viruses were assayed by the fluorescent antibody method in sera and cerebrospinal fluids (C.S.F.) obtained simultaneously from 30 patients with multiple sclerosis, 30 patients with other neurological diseases, and 30 “normal” control subjects. Sera of 11 out of 30 patients with multiple sclerosis had IgM which reacted specifically with measles virus-infected cells, compared with 2 out of 30 of the patients with other neurological diseases and none of the 30 normal controls. Virus-specific IgM was not found in C.S.F. by this method.The geometric mean titre of measles virus-specific IgG in serum was significantly higher in the multiple sclerosis group than in either control group, and while IgG specific for all three viruses was found in C.S.F., suggesting transfer across the blood-brain barrier, measles IgG predominated. 相似文献
2.
Pneumocystis carinii obtained from infected rat lung homogenates was incubated with fluorescein isothiocyanate-conjugated lectins, counterstained with the nuclear stain, propidium iodide (PI), and analyzed by dual parameter histograms for lectin-associated green and PI-associated red fluorescence using a fluorescence-activated cell sorter. The presence of glucose/mannose moieties was evidenced by the binding of all organisms to concanavalin A and Wisteria floribunda. From the lectin group specific for N-acetyl-D-glucosamine, P. carinii reacted strongly with wheat germ agglutinin and less intensely with Solanum tuberosum. Reaction with lectins specific for N-acetyl-D-galactosamine/galactose was variable, probably reflecting the secondary binding affinities of the lectins used. Soybean agglutinin, Bauhinia purpurea agglutinin, and Maclura pomifera agglutinin reacted moderately, whereas Dolichos biflorus agglutinin, and Griffonia simplicifolia I reacted less avidly. The organisms reacted partially with Ulex europaeus agglutinin, a lectin specific for fucose, and did not react well with Arachis hypogaea, Viscum album agglutinin, and Griffonia simplicifolia I beta 4, lectins specific for galactose. A very weak fluorescent signal was detected with Limax flavus agglutinin, suggesting little or no sialic acid was present. All lectin-binding reactions were confirmed for specificity by inhibition with the relevant carbohydrates. Flow cytometric analysis of lung-derived Pneumocystis organisms stained with fluorescent surface and nuclear dyes provides a rapid method for characterization of large parasite populations. 相似文献
3.
Production of Multivalent Fluorescent Antisera for Identification of Organisms in the Mycobacterium avium-Mycobacterium intracellulare Complex
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Antisera to ten strains of mycobacteria in the Mycobacterium avium-Mycobacterium intracellulare group were obtained by injecting rabbits with ultraviolet light-killed cells. The antisera were conjugated with fluorescein isothiocyanate and used in the direct fluorescent antibody test. Individual antisera reacted specifically with the mycobacterial serotype used to produce them. The antisera were then combined in two multivalent pools. Each multivalent pool reacted specifically with its corresponding antigens. The multivalent antisera were thus found to provide a rapid identification method for the mycobacteria studied. 相似文献
4.
A serological classification scheme for 48 strains of Bacteroides vulgatus was established by agglutination technique. Bacterial strains were isolated in our laboratory from gut flora of healthy persons. Absorbed antisera to 11 strains of B. vulgatus were prepared. All 48 strains tested reacted with one or more antisera. Among these strains, 22 serogroups were formed; 11 of them contained only one group component and 11 were made up of multiple group components. 相似文献
5.
The results of the cross reactions of the 27 strains of Azospirillum spp. with 4 fluorescent antibodies (FA) show a neat differentiation between the two species. A. lipoferum represents a more homogenous group in respect to FA reactions and highly fluorescent preparations were obtained with strains from a large scope origin against Sp59 FA, the type strain. In contrast A. brasilense contains at least three sub groups in respect to FA reactions. The first includes all denitrifing strains (nir+) which react with FA from Sp7 the type strain. None of the nir- strains reacted strongly with Sp7 FA. One part of the A. brasilense nir- group which includes the strains isolated from well sterilized rice and wheat roots (Sp 107, 107 st, 106 and 109 st) reacts with FA of their reference strain Sp107 but not with that of Sp28 FA. The strains isolated from unsterilized roots and soils reacted with SP28 FA and not with that of Sp107 FA. In addition there were 3 strains (Sp A4, 34 and 67) which reacted with neither of the FAs.Abbreviations Fa
fluorescent antibody
- FITC
fluorescein isothiocyanate
- Rh ITC
gelatin-rhodamine isothiocyanate
- nir+
nitrite reductase positive
- nir-
nitrite reductase negative 相似文献
6.
We report the site-specific fluorescent labeling of DNA using Staudinger ligation with high efficiency and high selectivity. An oligonucleotide modified at its 5' end by an azido group was selectively reacted with 5-[(N-(3'-diphenylphosphinyl-4'-methoxycarbonyl)phenylcarbonyl)aminoacetamido]fluorescein (Fam) under aqueous conditions to produce a Fam-labeled oligonucleotide with a high yield (approximately 90%). The fluorescent oligonucleotide was characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Because of the relatively high yield of the Staudinger ligation, simple purification of the product by size-exclusion chromatography and desalting is sufficient for the resulting fluorescent oligonucleotide to be used as a primer in a Sanger dideoxy sequencing reaction to produce fluorescent DNA extension fragments, which are analyzed by a fluorescent electrophoresis DNA sequencer. The results indicate that the Staudinger ligation can be used successfully and site-specifically to prepare fluorescent oligonucleotides to produce DNA sequencing products, which are detected with single base resolution in a capillary electrophoresis DNA sequencer using laser-induced fluorescence detection. 相似文献
7.
Development, Characterization, and Use of Monoclonal and Polyclonal Antibodies against the Myxosporean, Ceratomyxa shasta 总被引:5,自引:0,他引:5
J. L. BARTHOLOMEW J. S. ROHOVEC J. L. FRYER 《The Journal of eukaryotic microbiology》1989,36(4):397-401
Both monoclonal and polyclonal antisera were produced against Ceratomyxa shasta. Ascites containing trophozoites of the parasite was collected from infected fish and used as antigen for immunization of mice. The resulting monoclonal antibodies reacted specifically with trophozoite and sporoblast stages but did not react with C. shasta spores by either indirect fluorescent antibody techniques or in Western blots. This indicates that some C. shasta antigens are specific to certain life stages of the parasite. Polyclonal antiserum was produced in a rabbit by injecting a spore protein electro-eluted from an SDS-polyacrylamide gel. This antiserum reacted with both trophozoites and spores by indirect fluorescent antibody techniques and in Western blots. All antisera were tested for cross-reactivity to trout white blood cells, a contaminant of the ascites, and to other myxosporea. Two monoclonal antibodies reacted with white blood cells and myxosporea of the genera Sphaerospora and Myxobilatus. One hybridoma produced antibodies of high specificity for C. shasta pre-spore stages. This is the first report of a monoclonal antibody produced against a myxosporean parasite. 相似文献
8.
A novel, high-affinity, fluorescent progesterone receptor antagonist. Synthesis and in vitro studies
Hödl C Strauss WS Sailer R Seger C Steiner R Haslinger E Schramm HW 《Bioconjugate chemistry》2004,15(2):359-365
The present paper describes the chemical synthesis and in vitro characterization of a novel, high-affinity, fluorescent progesterone receptor (PR) antagonist. The three-step synthesis was carried out starting from mifepristone. After demethylation with calcium oxide, the methylamino group was alkylated with 6-bromohexanol, and the resulting compound was reacted with fluorescein 5-isothiocyanate, yielding the fluorescein-mifepristone conjugate. Interaction of the conjugate as well as of its precursors with PR was determined in cell culture (alkaline phosphatase assay and transactivation assay). Antiprogestagenic activity of the intermediates were comparable to that of the parent compound. Even after attachment of the bulky fluorescein moiety, considerable antiprogestagenic activity was maintained. Microscopic studies revealed that fluorescence of the conjugate was almost confined to the nuclei of steroid hormone receptor-positive cells, whereas the nuclei of steroid hormone receptor-negative cells remained unstained. To our knowledge, this is the first report on a fluorescent ligand for PR suitable for studies in living cells. It is proposed that the present fluorescent PR antagonist might serve as a lead compound for the development of contrast agents for PR imaging, e.g., by near-infrared optical imaging. 相似文献
9.
Serotypic diversity among Nitrobacter spp. isolates is greater than previously reported. Typing with fluorescent antibodies prepared against 11 Nitrobacter spp. cultures isolated from soil and water placed the isolates into six serogroups. When these fluorescent antibodies were applied to a group of 16 additional isolates, 8 were identifiable by cross-reaction to 3 of the 11 fluorescent antibodies. Some nitrite-oxidizing enrichment cultures from different habitats contained cross-reacting strains of Nitrobacter spp. 相似文献
10.
Neutralizing monoclonal antibodies to human rotavirus and indications of antigenic drift among strains from neonates. 总被引:37,自引:9,他引:28
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Cells producing neutralizing monoclonal antibodies to a serotype 3 human neonatal rotavirus strain RV-3 were derived by fusion of hyperimmunized mouse spleen cells with mouse myeloma cells. As ascites fluid, three rotavirus-neutralizing monoclonal antibodies were characterized by hemagglutination inhibition and reacted with 17 cultivable mammalian rotaviruses representing five virus serotypes, by fluorescent focus neutralization and enzyme immunoassay. Two antibodies, Mab RV-3:1 and Mab RV-3:2, reacted with the seven serotype 3 rotaviruses only. Mab RV-3:1 was shown to bind to the outer capsid glycoprotein gp34 of rotavirus when variants of SA 11 rotavirus were used, and it therefore appears to react with the major neutralization epitope of serotype 3 rotaviruses. The antibody Mab RV-3:3 was specific for an epitope of RV-3 rotavirus not present on any other rotavirus of any serotype tested, including another neonatal isolate of identical RNA electropherotype isolated from the same ward of the same hospital as RV-3 3 months earlier. These two viruses were also distinguishable by fluorescent focus neutralization, using antiserum to RV-3 virus. Western blot analysis showed binding of Mab RV-3:3 to the trypsin cleavage product of the outer capsid protein p86 of RV-3. This suggests that antigenic drift may have occurred among neonatal rotaviruses in Melbourne. These monoclonal antibodies will be useful in serotyping assays of rotaviruses directly in stool samples, and in further analysis of antigenic variation within the serotype. 相似文献
11.
12.
Characterization and localization of tropomyosin proteins in Xenopus embryos with specific antibodies 总被引:1,自引:1,他引:0
Tetsuya S. Tanaka Takashi Tatsuta Kohji Ikenishi 《Development, growth & differentiation》1995,37(1):111-122
In the process of monoclonal antibody (mAb) production against the 38kDa protein which is lacking in the gastrula arrested mutant embryos in Xenopus we incidentally obtained two kinds of mAb (designated as B11 and 2D10 antibodies, respectively) recognizing tropomyosin (TM) proteins in Xenopus embryos. The characterization of the corresponding antigens to those mAb was performed by immunoblotting and silver staining for two-dimensional (2-D) gels in the present study. The localization of the antigens in Xenopus embryos was also investigated by fluorescent microscopy.
By 2-D immunoblots with those mAb, three distinct protein spots or TM isoforms were recognized in Xenopus embryos; a 38 kDa spot with a pl of approximately 4.8 reacted with both antibodies in embryos at stages later than the mid-tailbud (stage 28) and two 30 kDa spots, which are probably isomers, with a pl of approximately 4.8 were detected with 2D10 antibody in embryos at stages extending from the fertilized to the mid-neurula (stage 20). By immunofluorescent microscopy, B11 antibody was shown to react mainly with muscle cells and their precursor cells. In contrast, 2D10 antibody stained the cytoplasm of almost all cells in embryos at stages from the fertilized to the tadpole.
Judging from the results obtained with immunoblotting and fluorescent microscopy, it is likely that the 38 kDa spot is a skeletal muscle TM isoform and the two 30 kDa spots are non-muscle TM isoforms. 相似文献
By 2-D immunoblots with those mAb, three distinct protein spots or TM isoforms were recognized in Xenopus embryos; a 38 kDa spot with a pl of approximately 4.8 reacted with both antibodies in embryos at stages later than the mid-tailbud (stage 28) and two 30 kDa spots, which are probably isomers, with a pl of approximately 4.8 were detected with 2D10 antibody in embryos at stages extending from the fertilized to the mid-neurula (stage 20). By immunofluorescent microscopy, B11 antibody was shown to react mainly with muscle cells and their precursor cells. In contrast, 2D10 antibody stained the cytoplasm of almost all cells in embryos at stages from the fertilized to the tadpole.
Judging from the results obtained with immunoblotting and fluorescent microscopy, it is likely that the 38 kDa spot is a skeletal muscle TM isoform and the two 30 kDa spots are non-muscle TM isoforms. 相似文献
13.
Cheng C. Yang John W. Fenton Richard D. Feinman 《Archives of biochemistry and biophysics》1981,208(2):610-614
Human α- and γ-thrombins were labeled with a fluorescent dansyl group, and tested for their utility in polarization of fluorescence experiments. The enzyme carbohydrate groups were oxidized with sodium periodate, labeled with dansyl-hydrazine, and reduced with sodium borohydride. Dansyl-α-thrombin showed 75% of the clotting activity of the unlabeled enzyme. The γ form (a proteolyzed derivative of α-thrombin with essentially no clotting activity) retained 90% of its esterase activity, and 60% of its amidase activity. Steady-state and polarization of fluorescence measurements of the two dansyl-thrombins were essentially the same indicating a similarity in gross structural properties despite the covalent modifications which convert the α to the γ form. Both dansyl-thrombins reacted with antithrombin and the expected decrease in tumbling time was observed in the fluorescence polarization measurements. High-affinity heparin had no effect on the fluorescence parameters for the dansyl-thrombin-antithrombin complex. Dansyl-α-thrombin also reacted with α2-macroglobulin; the polarization measurements indicated substantial immobilization suggesting the enzyme is not bound to subregions of high-rotational freedom. 相似文献
14.
Peripheral human lymphocytes reacted with fluorescein diacetate and analyzed by flow cytometry produced a bimodal fluorescence distribution that was shown to be attributable to the differential staining of T and B lymphocytes. Lymphocytes were fractionated into rosetting (T cell) and nonrosetting (B cell) populations. Both subfractions were reacted with fluorescein diacetate and analyzed by flow cytometry. The rosetting fraction was more fluorescent than the nonrosetting fraction, and the analysis of an appropriate mixture of the subfractionated populations produced a fluorescence distribution very similar to that obtained with unfractionated lymphocytes. 相似文献
15.
Mary E. Goodsell Julianna Toth John L. Johnson Dr. Cecil S. Cummins 《Current microbiology》1991,22(4):225-230
Of 38 strains ofPropionibacterium avidum, most fell into one of two groups. One group (20 strains) hadLl-diaminopimelic acid as the diaminoacid of peptidoglycan, did not ferment inositol, and reacted with serum to strain VPI 0576; the other group (11 strains) hadDl-diaminopimelic acid as the peptidoglycan diaminoacid, fermented inositol, and reacted with serum to strain VPI 0670. DNA sequence similarity studies showed that, while overall intergroup similarity was about 80%, within each group the sequence similarities were over 90%. Seven strains were anomalous and did not fit exactly into either group.The results show that the isomer of diaminopimelic acid in peptidoglycan may differ in strains of a single species all of which show at least 80% DNA sequence similarity to each other. 相似文献
16.
A M Martelli A M Billi C De Marchis L Manzoli L Cocco 《Cell biology international reports》1990,14(5):409-418
Rat liver nuclear matrices were reacted with the fluorescent dye 6-iodoacetamidofluorescein and the matrix proteins were then separated by one and two-dimensional polyacrylamide gel electrophoresis. Upon transillumination with U.V. light it was possible to see that several proteins had reacted with the dy, thus indicating the presence of free -SH groups. This labelling technique allowed the detection of a large number of proteins, being several folds more sensitive than conventional Coomassie Blue staining, as demonstrated by two-dimensional electrophoretical separation. If nuclear matrices were treated with reducing agents before being reacted with 6-iodoacetamidofluorescein, the fluorescence increased with about the same intensity in all the protein bands. It is proposed that 6-iodoacetamidofluorescein can be used as a specific and very sensitive probe to study the -SH groups of nuclear matrix proteins. 相似文献
17.
Sponge aggregation factor: in situ localization by fluorescent monoclonal antibody techniques 总被引:1,自引:0,他引:1
The aggregation factor (AF) from sponges mediates a heterophilic interaction of homologous cells. Applying electron microscopical means, we succeeded only very rarely in identifying the 90 S AF particle in tissue sections from Geodia cydonium. By means of a fluorescent antibody technique, we have now localized the cell binding domain of the AF in situ. Previous studies in this laboratory have led to the identification of the 47-kDa cell binding protein of the AF, using the monoclonal antibody (mab) 5D2-D11 [Gramzow M, Bachmann M, Zahn RK, Uhlenbruck G, Dorn A, Müller WEG, J Cell Biol, 102: 1344-1349, 1986]. This mab and mab 7D5, directed against a 92-kDa protein in the AF complex, were chosen for the fluorescent studies. By using mab 5D2-D11, the plasma membranes of cells from different regions in the sponge could be brightly stained. However, mab 7D5 reacted only very weakly with the sponge surfaces. By applying the immuno-blotting technique it was furthermore demonstrated that the cell binding protein is present both in the associated form with AF complex and in a free state. Moreover, it was established that the 47-kDa binding protein is not present in homologous glycoconjugates, lectin, or collagen; these components are known to be involved in cell-matrix interaction. 相似文献
18.
Three-color immunofluorescence histochemistry allowing triple labeling within a single section 总被引:1,自引:0,他引:1
W A Staines B Meister T Melander J I Nagy T H?kfelt 《The journal of histochemistry and cytochemistry》1988,36(2):145-151
We describe a procedure for simultaneous immunohistochemical localization of three different neuropeptides, neurotransmitters, or neurotransmitter enzymes within one and the same tissue section and present a number of examples of its application within the brain and periphery. Primary antibodies from three different species were bound to three different neurochemical substances within the same section and were then reacted with three appropriate species-specific antisera conjugated with fluorescein, rhodamine/Texas red, or biotin. The biotinylated secondary antiserum was subsequently reacted with diethylaminocoumarin (DAMC) conjugated to avidin. This combination resulted in green, red, and blue fluorescent labeling of each antigen, respectively. Each fluorescent marker was viewed and photographed discretely using appropriate excitation and suppression filter combinations. The method is well suited for analyzing instances of multiple coexistence at both the level of the cell soma and within terminal regions. More broadly, the feasibility of three-color immunofluorescence histochemistry extends the range with which antigen localization can be used to investigate the morphological bases of relationships and interactions between immunohistochemically characterized neuronal elements. 相似文献
19.
Antigenic differences between the cystozoite and endozoite of Toxoplasma gondii were found using fluorescent antibody staining. Antisera against the cystozoite reacted against only the cystozoite, whereas antisera against the endozoite reacted against both endozoite and cystozoite. Absorption of sera with endozoites removed only positive reactions with endozoites. These findings are the first to suggest antigenic differences between these two forms of Toxoplasma. 相似文献
20.
The structures of fluorescent products formed in the reaction of methyl linoleate hydroperoxides with adenine, FeSO4 and ascorbic acid were investigated to elucidate the mechanism of interaction. The fluorescent products consisted of at least four major components (I-IV), which could be separated by thin-layer chromatography and high-performance liquid chromatography. Both 2-octenal and 2,4-decadienal, degradation products of methyl linoleate hydroperoxides, reacted with adenine to produce a fluorescent product similar to one of the major compounds (II) formed in the reaction of methyl linoleate hydroperoxides. Spectroscopic data suggest that I and III are the same type of compounds, which have closed ring structures with alpha, beta-unsaturated carbonyl groups between the amino group at the 6-position and the nitrogen at the 1-position of adenine. Component II has a closed ring structure at the same site as I and III, and the presence of an ether linkage was suggested. On the basis of these structures, the involvement of 3-nonenal, methyl 12-oxo-9-dodecenoate and 2-octenal was suggested in the interaction of the methyl linoleate hydroperoxides decomposition products and adenine or DNA in the presence of FeSO4 and ascorbic acid. 相似文献