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1.
The alterations in the catalytic activity of the horseradish peroxidase after its interaction with antibodies against this enzyme have been studied in buffered solution and in reversed Aerosol OT (AOT) micelles in heptane. The antibodies were obtained by immunizing the rabbits with electrophoretically homogeneous enzyme and were purified by affinity chromatography. In the AOT micelles and mixed micelles containing AOT and Triton X-45, the enzyme interacted with antibodies very rapidly (in less than 5 min), i.e. the micelles did not hinder effective interaction between the enzyme and antibodies. The decrease in the peroxidase catalytic activity upon its interaction with antibodies in a micellar medium was determined by [H2O]/[AOT] ratio, pH and molarity of polar nucleus, as well as by the initial concentration of antibody. In buffered solutions, the decrease n the peroxidase activity of the enzyme--antibody complex was only weakly dependent on pH and molarity of a buffer solution. 相似文献
2.
On the basis of an epitope model, capture enzyme immunoassay systems using monoclonal antibodies have been devised for the detection and quantification of Tick-borne encephalitis virus and compared with a reference system employing polyclonal sera. Monoclonal antibodies were used both as capture and detector antibodies, their suitability depending primarily on their avidity and intrinsic background activity. A considerable increase in sensitivity was achieved by combining antibodies to different non-overlapping epitopes. Biotinylation of the detector antibodies allowed the construction of multiple site simultaneous binding assays. Furthermore the use of monoclonal antibodies of defined serological specificity made virus type identification possible. This assay can therefore be used as a rapid 'test of identity' as required during the manufacture of viral vaccines. 相似文献
3.
I L Degtiarev E I Shumakova S M Vorob'ev D A Kostiuk S N Kochetkov 《Molekuliarnaia genetika, mikrobiologiia i virusologiia》1989,(9):20-22
The monoclonal antibodies to DNA-dependent RNA-polymerase of bacteriophage T7 have been obtained. Twenty of the obtained 500 clones have inhibited the enzyme activity. Three specificity groups were identified for seven of the clones supporting their affinity for different antigenic determinants. 相似文献
4.
Matrix-bound monoclonal antibodies against urokinase have been used to purify this enzyme by affinity chromatography. In a single-step procedure, urokinase can be isolated from crude preparations with high yield and high purity, and without loss of enzymatic activity. 相似文献
5.
Virus surveillance and certification procedures for finfish have traditionally relied upon isolation of replicating agents in cell culture and identification using serological procedures. However, accurate monitoring may also be achieved using techniques to detect fish antibodies against viral disease agents. The serological procedures most used for detection of fish antibodies are the serum neutralization test and immunofluorescence. Other techniques such as enzyme linked immunosorbent assays (ELISA) have been less commonly used. Using infectious hematopoietic necrosis virus (IHNV) and viral hemorrhagic septicemia virus (VHSV) as examples, this paper reviews the serological test procedures used for rhabdoviral surveillance and the applications of this methodology to viral epizootiology and certification of finfish. 相似文献
6.
Methylmalonyl-CoA mutase, one of two known cobalamin-dependent enzymes present in mammalian tissues, has been isolated from 2.5 kg of human placenta utilizing affinity chromatography on 5'-deoxyadenosylcobalamin-Sepharose as the major purification step. The enzyme gives a single band on polyacrylamide disc gel electrophoresis. The Mr of the enzyme is 145,000 and it has two subunits of Mr = 72,000. Amino acid analysis reveals major differences from other human cobalamin-binding proteins. Based on x-ray fluorescence, the enzyme has 2 mol of cobalamin bound/mol of enzyme. In contrast to purified cobalamin transport proteins, most of the cobalamin bound to the enzyme is not released by boiling at low pH in the presence of KCN, or dialysis against 7.5 M guanidine containing 0.2 M dithiothreitol, or both, suggesting the possibility that cobalamin may be covalently attached to the purified enzyme. Both precipitating antibodies and antibodies that inhibit enzyme activity have been raised in a chicken. 相似文献
7.
N F Iankina T S Shobukhova I P Pavlova L B Bogoiavlenskaia T A Zykova 《Zhurnal mikrobiologii, epidemiologii, i immunobiologii》1989,(3):74-78
Obtaining antibodies to individual components of Salmonella antigenic complex is highly important for investigations aimed at the study of the antigenic structure of bacteria, their serological identification and the development of diagnostic preparations. The method of obtaining antibodies by the oxidation of Salmonella antigens with sodium periodate and creating immunosorbents based on these antibodies with subsequent affinity chromatography has been developed. Monospecific antibodies thus obtained (O2, O4, O9) have been studied and used as monospecific preparations in the agglutination test, the immunofluorescence test and the immunosorbent assay. The development of methods for stabilizing these preparations, thus ensuring their wide practical use, may be of interest. 相似文献
8.
Fractions of polar lipids have been isolated from bifidobacteria, and the immunoreactivity and serological specificity of glycolipids and phospholipids have been studied. Enzyme immunoassay (dot-EIA) of polar lipids demonstrates that the fractions of glycolipids and phospholipids of bifidobacteria are highly immunoreactive. Pronounced reactions of major glycolipids and phospholipids with a homologous polyvalent antiserum against B. adolescentis 94-BIM have been observed at antigen concentrations of approximately 100 ng. The antiserum contained high titers of specific antibodies against glycolipids and phospholipids of bifidobacteria, as demonstrated by heterogeneous solid-phase enzyme immunoassay (ELISA). Experimental data confirming the presence of subpopulations of specific antibodies (antiglycolipid and antiphospholipid) in the blood sera of immunized animals lead to the conclusion that the major glycolipids and phospholipids of bifidobacteria are specific markers appropriate for serological diagnostics. 相似文献
9.
Nuclear glutamate dehydrogenase: unique antigenic determinants and determinants common to the mitochondrial enzyme 总被引:1,自引:0,他引:1
An antiserum against glutamate dehydrogenase from ox liver nuclei precipitates both the nuclear and the mitochondrial enzymes, with different equivalence zones. The antibodies of this serum have been fractionated by means of an immunoadsorbent to which mitochondrial glutamate dehydrogenase is covalently linked. After the affinity chromatography, the unretained antibodies had virtually lost the ability to precipitate the mitochondrial enzyme, whereas the retained portion, after elution, precipitated both glutamate dehydrogenases. These findings suggest that nuclear glutamate dehydrogenase contains specific antigenic determinants as well as determinants common to the mitochondrial enzyme, and that only the antibodies against the latter determinants have been selectively removed by the affinity chromatography. 相似文献
10.
The beta subunit of E. coli glycyl-tRNA synthetase plays a major role in tRNA recognition.
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G M Nagel S Cumberledge M S Johnson E Petrella B H Weber 《Nucleic acids research》1984,12(10):4377-4384
The contributions made by the alpha and beta subunits of E. coli glycyl-tRNA synthetase to the recognition of tRNA have been investigated via binding and immunological methods. Using the nitrocellulose filter assay, we have shown that isolated beta subunit, but not the alpha subunit, binds [14C]glycyl-tRNA with an affinity comparable to that of the native enzyme. Further, the data indicate that the beta subunit possesses one binding site for glycyl-tRNA while the native or reconstituted enzyme (alpha 2 beta 2) has two sites. Rabbit antibodies directed at the beta subunit or the holoenzyme inhibit efficiently the ability of the enzyme to aminoacylate tRNA while alpha-subunit antibodies have a smaller effect. Since none of the antisera have an appreciable effect on the ATP-PPi exchange activity of the enzyme under these conditions, the beta-subunit (and holoenzyme) antisera evidently interfere with productive tRNA binding. Taken together, the data indicate that the larger, beta subunit of glycyl-tRNA synthetase plays a major role in tRNA recognition. 相似文献
11.
Fractions of polar lipids have been isolated from bifidobacteria, and the immunoreactivity and serological specificity of glycolipids and phospholipids have been studied. Enzyme immunoassay (dot-EIA) of polar lipids demonstrates that the fractions of glycolipids and phospholipids of bifidobacteria are highly immunoreactive. Pronounced reactions of major glycolipids and phospholipids with a homologous polyvalent antiserum against B. adolescentis 94-BIM have been observed at antigen concentrations of approximately 100 ng. The antiserum contained high titers of specific antibodies against glycolipids and phospholipids of bifidobacteria, as demonstrated by heterogeneous solid-phase enzyme immunoassay (ELISA). Experimental data confirming the presence of subpopulations of specific antibodies (antiglycolipid and antiphospholipid) in the blood sera of immunized animals lead to the conclusion that the major glycolipids and phospholipids of bifidobacteria are specific markers appropriate for serological diagnostics. 相似文献
12.
Isolation and characterization of 5'-nucleotidase of a human pancreatic tumor cell line 总被引:1,自引:0,他引:1
5'-Nucleotidase of a human pancreatic tumor cell line (PaTu II) has been purified to homogeneity after extraction with detergent followed by two affinity chromatographic steps. Sodium dodecyl sulphate polyacrylamide gel electrophoresis of purified 5'-nucleotidase revealed a single polypeptide band of 67 kDa. The Western blotted enzyme can be overlaid with concanavalin A proving its glycoprotein nature. After treatment with endoglycosidase F the deglycosylated 5'-nucleotidase exhibits an apparent molecular mass of 58 kDa. The kinetic properties of the solubilized enzyme have been determined (Km (AMP) of 4.0 microM; Vmax (AMP) = 8.6 muMOL/min.mg). Adenosine 5'-[alpha,beta-methylene]diphosphate is a competitive inhibitor of 5'-nucleotidase, whereas concanavalin A inhibits the enzymatic activity in a non-competitive manner. Polyclonal antibodies against purified 5'-nucleotidase of PaTu II have been produced which inhibit its enzymatic activity. Polyclonal antibodies raised against the enzyme purified from rat liver or bull seminal plasma also recognize 5'-nucleotidase of PaTu II cells, whereas polyclonal and monoclonal antibodies against the enzyme derived from chicken gizzard show no cross-reactivity. 5'-Nucleotidase appears to be concentrated in the plasma membrane of PaTu II cells as judged by cell fractionation and indirect immunofluorescence studies. 相似文献
13.
The effect of highly inhibitory rabbit antibodies on the enzymic activity of lobster-muscle arginine kinase has been studied. 100% inhibition was obtained at a 30-fold molar excess of antibody. The guanidine substrate L-arginine did not protect the enzyme from subsequent inhibition by its antibodies. On the other hand, the metal-nucleotide substrate Mg-ATP, or the substrate analog Mg-AMP, protected about 50% the enzyme activity, the extent of hte protection being irrespective of hte presence or of the absence of L-arginine and of the value of the molar ration Ab/Ag/ An antibody population directed against the antigenic determinant containing the essential tyrosine residue of the enzyme has been isolated by the affinity chromatography. No inhibitory acitivity was found in that fraction. Most of the inhibitory activity was found in the remaining antibody populations... 相似文献
14.
Numerous approaches to creating protein multilayers have been described for a variety of applications. In this work, the affinity of the enzyme pectinase towards a natural polysaccharide alginate has been exploited to create affinity directed enzyme multilayers on alginate beads. The biocatalyst has been characterized in terms of catalytic efficiency and kinetic parameters. Atomic force microscopy provided a good insight into multilayer formation at the molecular level. It was seen that enzyme layering was not uniform all over the bead. Thus, achieving high catalytic efficiency was an ‘average performance’ of the enzyme molecule, which was part of different layers. The results show that it is possible to create layered biocatalyst designs without using expensive lectins/antibodies for constructing affinity layers. 相似文献
15.
N V Tsvetkova Iu V Borisenko E M Kuznetsova L M Chudnaia E P Chaban 《Zhurnal mikrobiologii, epidemiologii, i immunobiologii》1987,(8):91-93
The sera of children and adults with a history of pertussis, as well as the sera of children immunized in three injections, have been studied in the enzyme immunoassay. The levels of antibodies to Bordetella pertussis protein and lipopolysaccharide, and to disintegrated B. pertussis cells have been determined; a serum titer of 1:1,600 and higher is considered as a criterion for the serological diagnosis of pertussis. 相似文献
16.
Charles H. Schmelzer Charlotte Phillips Harold E. Swaisgood H.Robert Horton 《Archives of biochemistry and biophysics》1984,228(2):681-685
A radioimmunoassay for sulfhydryl oxidase, a membrane enzyme, was developed using antibodies raised to the bovine milk enzyme which had been purified by transient covalent affinity chromatography on a cysteinylsuccinamidopropyl-glass matrix. Bovine milk sulfhydryl oxidase and bovine kidney sulfhydryl oxidase (“glutathione oxidase”) appear to be immunologically identical as evidenced by parallel responses in radioimmunoassays. Antibodies raised to the purified milk sulfhydryl oxidase can immunoprecipitate glutathione oxidase activity, but not γ-glutamyltransferase (“transpeptidase”) activity, from bovine kidney preparations. 相似文献
17.
Two previously obtained, full-size, fully human antibodies that reversibly bind the active form of an enzyme belonging to the subtype EC 3.2.1, which is used for substitutive enzymatic therapy in lysosomal storage diseases, have been characterized by surface plasmon resonance and biolayer interferometry methods. It was shown under normal physiological conditions that the designed antibodies specifically bound with the antigen (KD ~ 10–8 M) and rapidly dissociated at neutral pH in 60% ethylene glycol while leaving the enzymatic activity unchanged. Dan ue to their properties, the developed antibodies can be used in industry as affinity ligand in the isolation of therapeutic substances of the enzyme. 相似文献
18.
D Iu Plaskin G A Ermolin L K Shipina M A Vladimirski? 《Zhurnal mikrobiologii, epidemiologii, i immunobiologii》1988,(8):79-82
A solid-phase enzyme immunoassay system for the determination of antibodies to mycobacterial antigens, based on the method of erythrocyte immunoadsorption in microchambers for immunological reactions, has been developed. To detect antibodies specifically bound with the solid-phase antigen, the affinity rosettes of Staphylococcus aureus strain Cowan I, carrying protein A, with erythrocytes conjugated with human gamma globulin have been used. The significant correlation of the titers of 34 sera, determined by means of erythrocyte immunoadsorption, with extinction values obtained in the solid-phase enzyme immunoassay of antibodies to Mycobacterium tuberculosis has been established. The coincidence of the results in 92% of cases has been noted. 相似文献
19.
Affinity purification and refined structural characterization of terminal deoxynucleotidyltransferase.
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A total of 56 stable murine hybridoma monoclones that produce homogeneous antibodies against human or calf terminal deoxynucleotidyltransferase have been established. All of the antibodies exhibited specific binding to various Mr forms of terminal transferase and eight possessed neutralizing activity. Results are presented that permitted characterization of ten of these antibodies with respect to their immunoglobulin class, their recognition of calf or human terminal-transferase Mr species by immunoblotting techniques and their recognition of distinct antigenic sites. Terminal transferase was purified in a single step by using an immunoaffinity column constructed with a monoclonal antibody exhibiting a high binding affinity for the enzyme. Single monoclonal antibodies were also used to bind selectively to terminal-transferase antigen in tissue slices and individual cells. 相似文献
20.
Biochemical and immunological characterization of the STA2-encoded extracellular glucoamylase from saccharomyces diastaticus 总被引:1,自引:0,他引:1
In Saccharomyces diastaticus each one of three unlinked genes (STA1, STA2, STA3) encodes a glucoamylase (alpha-1,4 glucanglucohydrolase, EC 3.2.1.3) that allows yeast to grow on starch. The enzyme encoded by the STA2 gene (glucoamylase II) has been purified from culture medium to near homogeneity by ethanol precipitation, Trisacryl M DEAE chromatography, and HPLC gel filtration. Glucoamylase II consists of two identical subunits whose average size is 300 kDa. Under denaturing conditions, the native dimeric enzyme readily dissociates to a monomer. Enzymatic deglycosylation of denatured enzyme gives rise to intermediate, partially glycosylated forms and to a 56-kDa completely deglycosylated protein. Glucoamylase releases glucose units by cleaving alpha-1,4 bonds from the nonreducing end of different oligosaccharides, but has only a barely detectable alpha-1,6 hydrolyzing activity. The pH optimum for the purified enzyme was found to be 5.1. The enzyme has a greater affinity for maltohexaose (Km = 0.98 mM, V/Km = 2.39) than for maltotriose (Km = 2.38, V/Km = 0.68) or maltose (Km = 3.20, V/Km = 0.39). Both polyclonal and monoclonal antibodies have been raised against glucoamylase II. The polyclonal antibodies specifically inhibit yeast glucoamylase II activity in a dose-dependent manner, but are found to immunoblot other yeast glycoproteins as well. This oligosaccharide-specific reaction can be competed out by adding excess mannan without affecting glucoamylase reactivity. The cross-reactivity of the polyclonal antibodies with other amylolytic enzymes correlates well with evolutionary distance. Evidence is presented that monoclonal antibodies specific for either carbohydrate or protein epitopes have been obtained. 相似文献