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1.
Antibodies to Escherichia coli translational initiation factors IF2 and IF3 were used for an immunological comparison of unpurified proteins from the following genera: Salmonella, Serratia, Proteus, Aeromonas, Pseudomonas, Streptococcus, Sarcina and Bacillus. Immunological relatedness was compared by Ouchterlony double diffusion experiments and immunoblotting analysis. Immunoblotting is a quantitative technique for measuring levels of specific proteins in crude cell lysates. We have used this technique to measure immunological distance with the assumption that the levels of the various translational components are essentially the same in the different bacterial cells examined. Both immunodiffusion and immunoblotting analysis showed a similar evolutionary relationship between the various species for the two initiation factors examined: (Escherichia=Salmonella>Serratia>Proteus>Aeromonas>Pseudomonas). Little or no crossreactivity was found using either analysis with genera: Streptococcus, Sarcina and Bacillus. Using the immunoblot distance, the two initiation factors were shown to diverge at similar rates. One advantage the immunoblotting analysis has over other immunological techniques is that the antigens can be analyzed structurally. We found, for example, that the two forms of IF2 were present in all bacterial species which cross-reacted with anti-IF2, suggesting that both forms are functionally important. Because of its sensitivity, the immunoblot analysis may be more useful than other immunological techniques in studying species that are more distantly related.Abbreviations used IF
initiation factor
- MOPS
morpholinopropane sulfonate
- PAGE/SDS
polyacrylamide gel electrophoresis/sodium dodecyl sulfate 相似文献
2.
Non-Immunological Precipitation of Serum by Sodium Dodecyl Sulfate in Agar Diffusion 总被引:3,自引:1,他引:2
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Marmoset serum or serum of other species of animal may react with sodium dodecyl sulfate and forms nonspecific precipitin lines in agar diffusion. The protein detergent complexes are not readily dialyzable. Therefore precipitin lines derived from studies that use sodium dodecyl sulfate-treated antigens in agar diffusion must be interpreted with caution. 相似文献
3.
The MIC of 1,10-phenanthroline for 35 Pseudomonas aeruginosa strains was 128 micrograms/ml, whereas 32 micrograms or less per ml inhibited all other microorganisms tested. On the basis of these results, a selective agar for P. aeruginosa which contained 15 g of Trypticase soy broth (BBL Microbiology Systems), 15 g of agar, and 0.1 g of phenanthroline per liter was formulated. Forty-four P. aeruginosa strains yielded a mean efficiency of plating on this medium of 79% of the counts obtained on Trypticase soy agar, which was significantly higher than that obtained with pseudomonas isolation agar or Pseudosel agar. Pseudomonas cepacia, Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas stutzeri, representatives of 13 other genera (including gram-negative rods, gram-positive rods, and cocci), and a yeast were not recovered within 48 h at 35 degrees C when approximately 10(7) CFU were plated on this medium. Only small colonies from one strain each of P. fluorescens and P. putida could be seen at 3 and 7 days, respectively, and they had an efficiency of plating of only less than 0.001%. When 10(7) CFU of either of these strains was plated with 10(2) CFU of P. aeruginosa, it did not interfere with the quantitative recovery of P. aeruginosa. 相似文献
4.
Selective medium for Pseudomonas aeruginosa that uses 1,10-phenanthroline as the selective agent. 总被引:2,自引:2,他引:0
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The MIC of 1,10-phenanthroline for 35 Pseudomonas aeruginosa strains was 128 micrograms/ml, whereas 32 micrograms or less per ml inhibited all other microorganisms tested. On the basis of these results, a selective agar for P. aeruginosa which contained 15 g of Trypticase soy broth (BBL Microbiology Systems), 15 g of agar, and 0.1 g of phenanthroline per liter was formulated. Forty-four P. aeruginosa strains yielded a mean efficiency of plating on this medium of 79% of the counts obtained on Trypticase soy agar, which was significantly higher than that obtained with pseudomonas isolation agar or Pseudosel agar. Pseudomonas cepacia, Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas stutzeri, representatives of 13 other genera (including gram-negative rods, gram-positive rods, and cocci), and a yeast were not recovered within 48 h at 35 degrees C when approximately 10(7) CFU were plated on this medium. Only small colonies from one strain each of P. fluorescens and P. putida could be seen at 3 and 7 days, respectively, and they had an efficiency of plating of only less than 0.001%. When 10(7) CFU of either of these strains was plated with 10(2) CFU of P. aeruginosa, it did not interfere with the quantitative recovery of P. aeruginosa. 相似文献
5.
ANTIGENS IN EGGS AND DEVELOPMENTAL STAGES OF THE SEA URCHIN : I. Immunological and Physicochemical Properties 总被引:1,自引:1,他引:0
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A number of antigens in unfertilized eggs and embryos of the sea urchin Paracentrotus lividus were characterized with respect to both immunological and physicochemical properties. Experiments involved single diffusion in agar (Oudin technique) combined with mutual dilution, serial dilution, and heating of antigenic extracts, as well as immunoelectrophoresis with normal and heated extracts and agar electrophoresis followed by staining of the antigenic spots with protein specific dyes. The gradual transition in migration rates of bands of precipitates in Oudin tubes following mutual dilution of either extracts or antisera allowed the identification of 6 immunologically identical antigens in eggs and embryonic stages. Similarities with respect to diffusion coefficients, sensitivity to heat, electrophoretic mobility, and reaction to protein specific dyes indicated that the antigens in extracts of eggs and various developmental stages also had certain physicochemical properties in common. Such knowledge is of importance for an understanding of antigenic changes occurring during ontogenesis. 相似文献
6.
Detection of polysaccharide, teichoic acid, and protein antigens in bacterial colonies on an agar surface 总被引:1,自引:0,他引:1
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An improved method of using fluorescein-labeled antibody for the detection of polysaccharide, protein, and teichoic acid antigens synthesized by streptococcal colonies on an agar surface is described. The bacteria were grown on the surface of an agar medium contained in the shallow well of an immunodiffusion slide. An agar overlay containing the fluorescein antiserum was dispensed over the colonies, excess antiserum was washed out of the overlay agar, and the fluorescent colonies were observed under an ultraviolet microscope. The shallow well in the immunodiffusion slide prevented the agar from floating loose during washing, and the agar overlay prevented the fragmentation and loss of colonies. The thin layer of agar facilitated microscopic examination and the counting of fluorescent and nonfluorescent colonies. Colonies producing an antigen against which the antiserum was directed could readily be distinguished from colonies not producing the antigen. The specificity of the method was shown by using mixtures of streptococci representing six serological groups and five types. Those not known to possess cross-reacting antigens were specific in their reaction to the fluorescein antibody. Cross-reactions between the group antigens of A, C, and G, as reported previously by fluorescent staining of streptococcal suspensions, were also seen. Group A colonies reacted weakly with fluorescent E antibody and vice versa. The extraction of this antigen with cold trichloroacetic acid indicates it was related to the teichoic acids. Colonies possessing polysaccharide, protein, and teichoic acid antigens gave equally strong fluorescent reactions. This procedure permits detection of the synthesis of antigen which could not be observed by the use of a selective medium; it also eliminates the necessity for subculture of each colony and testing by appropriate serological means. Such a technique has value for studies in classification and biochemical genetics, and should be applicable to other genera of bacteria. 相似文献
7.
AUTORADIOGRAPHIC ANALYSIS ON AGAR PLATES OF ANTIGENS FROM SUB CELLULAR FRACTIONS OF RAT LIVER SLICES
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Slices of rat livers were incubated with 14C amino acids, homogenized, and subjected to differential centrifugation. The microsomes were further extracted with the non-ionic detergent Lubrol W and with EDTA. These extracts and the microsome free "cell sap," freed from the pH 5 precipitable fraction, were subsequently reacted with antisera using agar diffusion techniques. The antisera employed were obtained from rabbits injected with different subcellular fractions of rat liver or with rat serum proteins. When the agar diffusion plates were autoradiographed it was found that some of the precipitates were radioactive while others were not. Control experiments indicated that this labeling was due to the specific incorporation of 14C amino acids into various rat liver antigens during incubation of the slices rather than to a non-specific adsorption of radioactive material to the immunological precipitates. When the slices were incubated with the isotope for up to 30 minutes, the serum proteins which could be extracted from the microsomes with the detergent were strongly labeled, as were a number of additional microsomal antigens of unknown significance. In contrast, the serum proteins present in the cell sap were only weakly labeled. Most of the typical cell sap proteins, both those precipitable and those soluble at pH 5, seemed to remain unlabeled. No consistently reproducible results were obtained with the EDTA extracts of the ribosomal residues remaining after extraction of the microsomes with the detergent. Incubation of the liver slices for longer periods (up to 120 minutes) led to a strong labeling of the serum proteins in the cell sap as well as to the appearance of labeling in additional cell sap proteins. The results are discussed with regard to the subcellular site of synthesis and the metabolism of the different antigens. 相似文献
8.
W A Bi? 《Medycyna do?wiadczalna i mikrobiologia》1990,42(3-4):156-162
The aim of this study was to determine the occurrence of antibodies against antigens of A. flavus (APP, AEM, AS, API), A. fumigatus and A. candidus. One hundred and fifty two sera of individuals connected with industrial environment were tested, in which A. flavus was permanently isolated: 339 sera of healthy controls-blood donors of city of Poznań, and 24 sera of patients with confirmed or suspected Aspergillosis were also included in the study. The sera were tested for a presence of specific antibodies by immunoprecipitation in 1% agar gel, by using inactivated sera and above mentioned antigens. In a group of people having permanent contact with A. flavus, antibodies to antigens derived from this genus were present in 4.6% of individuals while against A. fumigatus antigens in 0% and A. candidus 0.7%. In blood donors group 5 times lower percentage of sera having anti-A. flavus antibodies was found and a complete lack of detectable antibodies for other two genera. The results of the studies of patient sera indicate a necessity of broadening a set of fungal antigens used for an investigation of this type of sera. Antibodies against A. flavus were found in three patients and for A. fumigatus in 7 patients. One patient had antibodies for both genera and two patients had antibodies against A. flavus lacking antibodies against A. fumigatus. The results of this study indicate that antigens of A. flavus should be included into serodiagnosis of Aspergillosis. 相似文献
9.
《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1995,663(1):51-57
A gel electrophoretic method coupled with agar diffusion has been devised for detecting tumor antigens in human colon tissue. Separation of the antigens is achieved on duplicate electrophoretic gels. One gel is used for the location of the antigens by protein staining and the other gel is used for assaying of the antigenicity by agar diffusion against homologous antiserum. Analysis of perchloric acid extracts of colon tumors by this coupled method revealed the presence of carcinoembryonic antigen and two additional glycoprotein antigens. Analysis of KClHCl tumor extracts revealed two new tumor antigens. 相似文献
10.
Outer Membrane Protein Heterogeneity within Pseudomonas fluorescens and P. putida and Use of an OprF Antibody as a Probe for rRNA Homology Group I Pseudomonads
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The electrophoretic patterns of outer membrane proteins of strains representing the biovars of Pseudomonas fluorescens and Pseudomonas putida were analyzed by gel electrophoresis. The outer membrane protein profiles were variable, and they were not useful for assigning strains to a specific biovar. However, three or four predominant outer membrane proteins migrating at 42 to 46 kDa, 33 to 38 kDa, and 20 to 22 kDa were conserved among the strains. They could be tentatively identified as OprE (44 kDa), OprF (38 kDa), OprH (21 kDa), and OprL (20.5 kDa), which are known proteins from Pseudomonas aeruginosa. A 37-kDa OprF-like protein was purified from P. fluorescens DF57 and used to raise a polyclonal antibody. In Western blot (immunoblot) analysis, this antibody reacted with OprF proteins from members of Pseudomonas rRNA homology group I but not with proteins from nonpseudomonads. The heterogeneity in M(infr) of OprF was greater among P. fluorescens strains than among P. putida strains. Immunofluorescence microscopy of intact cells demonstrated that the antibody recognized epitopes that were accessible only after unmasking by EDTA treatment. The antibody was used in a colony blotting assay to determine the percentage of rRNA homology group I pseudomonads among bacteria from the rhizosphere of barley. The bacteria were isolated on 10% tryptic soy agar, King's B agar, and the pseudomonad-specific medium Gould S1 agar. The estimate of OprF-containing CFU in rhizosphere soil obtained by colony blotting on 10% tryptic soy agar was about 2 and 14 times higher than the values obtained from King's agar and Gould S1 agar, respectively, indicating that not all fluorescent pseudomonads are scored on more specific media. The colonies reacting with the OprF antibody were verified as being rRNA homology group I pseudomonads by using the API 20NE system. 相似文献
11.
Surface Antigens of Smooth Brucellae 总被引:29,自引:2,他引:27
Surface antigens of smooth brucellae were extracted by ether-water, phenol-water, trichloroacetic acid, and saline and examined by immunoelectrophoresis and gel diffusion with antisera from infected and immunized rabbits. Ether-water extracts of Brucella melitensis contained a lipopolysaccharide protein component, which was specific for the surface of smooth brucellae and was correlated with the M agglutinogen of Wilson and Miles, a polysaccharide protein component devoid of lipid which was not restricted to the surface of smooth brucellae and was not correlated with the smooth agglutinogen (component 1), and several protein components which were associated with internal antigens of rough and smooth brucellae. Immunoelectrophoretic analysis of ether-water extracts of B. abortus revealed only two components, a lipopolysaccharide protein component, which was correlated with the A agglutinogen, and component 1. Component 1 from B. melitensis and B. abortus showed identity in gel diffusion tests, whereas component M from B. melitensis and component A from B. abortus showed partial identity with unabsorbed antisera and no cross-reactions with monospecific sera. Attempts to prepare monospecific sera directly by immunization of rabbits with cell walls or ether-water extracts were unsuccessful. Absorption of antisera with heavy fraction of ether-water extracts did not always result in monospecific sera. It was concluded (as has been described before) that the A and M antigens are present on a single antigenic complex, in different proportions depending upon the species and biotype, and that this component is a lipopolysaccharide protein complex of high molecular weight that diffuses poorly through agar gel. Components 1, A, and M were also demonstrated in trichloroacetic acid and phenol-water extracts. With all extracts, B. melitensis antigen showed greater diffusibility in agar than B. abortus antigens. After mild acid hydrolysis, B. abortus ether-water extract was able to diffuse more readily. 相似文献
12.
Bratislava mosaic virus was transmitted mechanically toChenopodium quinoa. The virus was isolated by means of gradient ultracentrifugation and the, proteins were distributed on an automatic recording photometer. Single fractions were taken with a fraction collector and used as antigen. At the same time, antigen from leaves of the variety Sylván zelený infected with the Bratislava mosaic virus was prepared by means of the gradient ultracentrifugation. The antisera were obtained from rabbits immunized with individual antigens. The antisera were tested with the saps ofC. quinoa infected with the Bratislava mosaic virus, by the method of double diffusion into agar according to Ouchterlony, and with adjusted saps from suckers infected with the Bratislava mosaic virus, by the method of double diffusion into agar according to Oudin. A specific reaction can be obtained in both cases only under the assumption that all conditions mentioned in the study are strictly kept. 相似文献
13.
Studies on the Mitotic Apparatus of the Sea Urchin by Means of Antigen-Antibody Reactions in Agar 总被引:2,自引:3,他引:2
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Hans A. Went 《The Journal of cell biology》1959,6(3):447-455
The primary purpose of the experiments reported in this paper was to gain information on the molecular origin of the mitotic apparatus. Antisera were prepared against unfertilized sea urchin (Strongylocentrotus purpuratus) egg antigens and mitotic apparatus antigens. These were permitted to react with various antigen solutions in Ouchterlony agar gel diffusion plates, and the resultant precipitation patterns analysed. The results revealed that the mitotic apparatus contains probably no more than two antigens (precursor-1 component and precursor-2 component) and that these are shared by the unfertilized egg. Absorption and fractionation techniques indicated that in the unfertilized egg the precursor-1 component is present both as a "soluble" protein and as an insoluble form tenaciously associated with intracellular structural elements. A survey of dividing and non-dividing tissues for the precursor-1 component revealed that it was restricted to tissues in which mitotic activity could be detected microscopically. No immunochemical relationship could be detected between the mitotic apparatus and proteins extracted, by various methods, from the lantern muscle. 相似文献
14.
IMMUNOLOGICAL STUDIES OF ISOLATED PARTICULATES OF PARAMECIUM AURELIA : I. Antigenic Relationships Between Cytoplasmic Organelles and Evidence for Mitochondrial Variations as Demonstrated by Gel Diffusion 总被引:1,自引:0,他引:1
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Irving Finger Michael Kaback Philip Kittner Carol Heller 《The Journal of cell biology》1960,8(3):591-601
Mitochondria and other particulates—cilia, trichocysts, and "small granules"—have been isolated from several stocks of Paramecium aurelia, syngen 2. Antisera against these particles and against breis have been used to characterize the fractions by diffusion in gel. Evidence is presented for the relationship of particles, as demonstrated by immunologic cross-reactivity of the soluble antigens extracted from them. Although some antigens are unique for a fraction, cross-reacting antigens in two or more fractions, as determined by "spur" formation in agar, suggest a relationship between morphologically diverse particles. A procedure for studying cross-reactions in gels is described using the specific immobilization antigens as a model. The localization of these antigens within cilia, and perhaps trichocysts, has been confirmed. Other organelles, specifically mitochondria and "small granules," appear to alter their specificity spontaneously and reversibly during cell reproduction, a pattern reminiscent of the immobilization serotypes which can transform to one another during clonal growth. 相似文献
15.
Differences Between Brucella Antigens Involved in Indirect Hemagglutination Tests with Normal and Tanned Red Blood Cells 总被引:5,自引:2,他引:3
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Brucella antigens capable of sensitizing normal and tanned sheep red blood cells for indirect hemagglutination were compared with antigens involved in agglutination, gel diffusion, and immunoelectrophoresis. Hyperimmune rabbit sera, before and after absorption with various antigenic preparations from smooth and rough B. abortus, were used in the tests. Normal erythrocytes could be sensitized with an NaOH-treated ether-water extract (EW-T) of smooth Brucella. Tanned erythrocytes could be sensitized with a water-soluble extract from ultrasonically disrupted smooth or rough Brucella. The EW-T produced a single precipitation band and the water-soluble antigens produce 6 to 23 bands in immunoelectrophoresis with unabsorbed sera. After absorption of antisera with water-soluble extracts from smooth or rough Brucella cells or from smooth or rough cell walls, the hemagglutinins for sensitized tanned erythrocytes and the precipitins for water-soluble antigens were removed. Absorption with living smooth or rough Brucella cells or with EW-T did not remove these antibodies. The precipitins and hemagglutinins for the antigen EW-T, and agglutinins for smooth cells, were absorbed by smooth antigens but not by rough antigens. It appears that the antigens which sensitize tanned erythrocytes and diffuse through agar gels are present in both smooth and rough forms and may be situated in the cytoplasm or in the internal part of the cell wall, whereas the agglutinogen and the antigen which attaches to normal erythrocytes are surface antigens found only on the smooth Brucella cell. 相似文献
16.
Preliminary experiments indicate that serological techniques provide a useful method of identifying specific prey antigens from the stomach contents of fish predators. Problems with cross-reacting antigens from different prey species can be reduced by absorbing anti-sera with tissue extracts of the cross-reacting species. Very small volumes of part-digested prey tissue are sufficient for identification of the prey species when using agarose diffusion plates (Ouchterlony tests). 相似文献
17.
The ABAC system for antibiotic susceptibility testing was compared with an agar diffusion method in 14960 tests, including 23 antibacterial agents. Identical breakpoints were used. Only 3% major discrepancies (M.d.; sensitive vs resistant) and 19% minor discrepancies (m.d.; intermediate vs sensitive or resistant) were noted. Major discrepancies were mainly found for methicillin ( Staphylococcus aureus ), netilmicin ( Pseudomonas aeruginosa ), chloramphenicol, sulphamethoxazole and tri-methoprim ( Proteus sp.) and were checked by quantitative susceptibility tests. These showed ABAC to be at fault in 41–47% of discrepancies, the diffusion test in 21–32% and 21–37% were intermediate. Half of the m.d. involved beta-lactams, which is explained by too low breakpoints. Except for methicillin and netilmicin the overall results showed ABAC to be equal to the agar diffusion method. Technical faults, like leakage and incorrect filling of cups in the plastic rotors of ABAC, occurred in 14% of the rotors. 相似文献
18.
Fedorov AIu Volchenko EV Singirtsev IN Korzhenevich VI Shub GM 《Prikladnaia biokhimiia i mikrobiologiia》2000,36(1):59-67
Our study of the techniques of long-term storage of the biomass of various strains of microorganisms, which cause breakdown or transformation of synthetic organic compounds, demonstrates that desiccated agar beads with immobilized microbial cells can be used for this purpose. In addition, the cells can be stored in desiccated matrices of agar or polyvinyl alcohol, coating synthetic cords. Such dry biocatalysts may be used for quick starting of bioreactors and in other biotechnological processes. The technique is applicable to storage of various strains of Pseudomonas, Corynebacterium, Rhodococcus, and, to a lesser extent, Enterobacteriaceae. 相似文献
19.
Silva MT Simas SM Batista TG Cardarelli P Tomassini TC 《Memórias do Instituto Oswaldo Cruz》2005,100(7):779-782
Complex physalin metabolites present in the capsules of the fruit of Physalis angulata L. have been isolated and submitted to a series of assays of antimicrobial activity against Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 29213, S. aureus ATCC 25923, S. aureus ATCC 6538P, Neisseria gonorrhoeae ATCC 49226, Escherichia coli ATCC 8739; E. coli ATCC 25922, Candida albicans ATCC 10231 applying different methodologies such as: bioautography, dilution broth, dilution agar, and agar diffusion techniques. A mixture of physalins (pool) containing physalins B, D, F, G inhibit S. aureus ATCC 29213, S. aureus ATCC 25923, S. aureus ATCC 6538P, and N. gonorrhoeae ATCC 49226 at a concentration of 200 mg/microl, using agar dilution assays. The mixture was inactive against P. aeruginosa ATCC27853, E. coli ATCC 8739; E. coli ATCC 25922, C. albicans ATCC 10231 when applying bioautography assays. Physalin B (200 microg/ml) by the agar diffusion assay inhibited S. aureus ATCC 6538P by +/- 85%; and may be considered responsible for the antimicrobial activity. 相似文献
20.
It was established by agar immunoelectrophoresis that Salmonella paratyphi B lysate contains a large number of soluble antigens which display a varying degree of serological specifity as well as different diffusion and electrophoretic mobility. Salmonella paratyphi B was found to possess, apart from specific O and H antigens, a surface K antigen. This is a distinct antigen having strict serological specificity. Purified K antigen displayed anodic mobility in immunoelectrophoresis. A detailed study of K antigen properties in cultures treated by different methods as well as immunochemical investigations of purified K antigen showed that the surface K antigen of S. paratyphi B differs from its O, M, Vi, H and other known antigens in terms of basic characteristics. 相似文献