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1.
Monoclonal antibodies designated as Am1 and Am2 were prepared against purified beef pancreas tryptophanyl-tRNA synthetase (EC 6.1.1.2). Both antibodies were able to bind the native enzyme in a solid-phase assay and to precipitate enzyme activity in immune complexes. Am2 inhibited the tryptophanyl-tRNA synthetase activity in ATP-[32P]pyrophosphate exchange and in tRNATrp aminoacylation reactions; Am1 had no influence on both the enzyme activity and the inhibiting action of Am2. Only Am2, not Am1, bound elastase-modified form of the enzyme which consists of two subunits shortened by 20 000 daltons from the N-end of the molecule. These results were interpreted as an evidence for non-overlapping of Am1 and Am2 antigenic determinants along the polypeptide chains of the enzyme. 相似文献
2.
Monoclonal antibodies were produced to electrophoretically pure hydrogenase fromThiocapsa roseopersicina. Protein immunoelectroblotting was used to identify the hydrogenase-specific antibodies. Among the 18 monoclonal antibodies selected by enzyme immunoassay, three were found to react with highly immunogenic trace contaminating proteins. One cell line produced antibody that inhibitied hydrogenase activity. This was the first specific inhibitor of the hydrogenase function. The results suggest that monoclonal antibodies could provide valuable new informations about the enzyme structure as well. 相似文献
3.
Production of monoclonal antibodies against avidin 总被引:1,自引:0,他引:1
R Ashorn P Ashorn M Kulomaa P Tuohimaa K Krohn 《Comparative biochemistry and physiology. B, Comparative biochemistry》1985,82(1):123-127
Monoclonal antibodies of the IgG1 subclass were generated against chicken avidin. These antibodies were shown to be as sensitive as polyclonal antiserum in detecting avidin by radioimmunoassay (RIA) and enzyme linked immunosorbent assay (ELISA) methods. Furthermore, the monoclonal antibodies were considerably more specific. Our results with a monoclonal anti-avidin RIA support previous findings that in inflammatory conditions avidin is synthesized also in other organs than the oviduct, although in the liver a major part of the activity detected by polyclonal anti-avidin RIA or biotin-bentonite assay was not due to avidin. 相似文献
4.
The development of a solid-phase immunosorbent assay, suitable for use with enzyme antigens, is described. Acid sphingomyelinase and a mouse monoclonal anti-sphingomyelinase antibody have been used to determine optimal conditions for the assay. The assay involves immobilization of a second antibody (anti-mouse IgG) in the wells of a polyvinyl microtiter plate. Soluble immune complexes of first antibody (monoclonal anti-sphingomyelinase) and antigen (sphingomyelinase), incubated in separate vials, are then reacted in the anti-mouse IgG-coated assay wells, and the extent of the cross-reaction between antibody and antigen is measured by direct assay of enzyme retained in the well. A necessary condition of the assay is that antibody must not inhibit enzyme activity, which makes it especially suitable for monoclonal antibodies. The assay finds useful application in hybridoma fluid screening, equivalence point determination, and demonstration of cross-reacting enzyme from various tissue sources. 相似文献
5.
Monoclonal antibodies against salt-resistant rat liver lipase. Cross-reactivity with lipases from rat adrenals and ovaries 总被引:1,自引:0,他引:1
To obtain monoclonal antibodies against rat salt-resistant liver lipase, mice were immunized with enzyme purified from heparin-containing rat liver perfusates. Hybridomas were screened for antibody production by means of an enzyme-linked immunosorbent assay (ELISA) and an immunoprecipitation assay. Five hybridoma cell lines secreting antibodies against rat liver lipase indicated as A, B, C, D and E, have been obtained. All antibodies possess gamma one (gamma 1) heavy chains and kappa (kappa) light chains. The antibodies precipitate salt-resistant lipase from rat post-heparin plasma, are positive in ELISA, inhibit liver lipase activity and bind monospecifically with the enzyme as shown by immunoblotting. The monoclonal antibodies showed no significant reactivity with human liver lipase. The salt-resistant lipases of rat adrenals and ovaries are also precipitated by the monoclonal antibodies directed against the liver enzyme. Therefore, the heparin-releasable lipases of the liver, adrenals and ovaries possess identical epitopes. 相似文献
6.
Emanuel PA Dang J Gebhardt JS Aldrich J Garber EA Kulaga H Stopa P Valdes JJ Dion-Schultz A 《Biosensors & bioelectronics》2000,14(10-11):751-759
Antibodies are critical reagents used in several biodetection platforms for the identification of biological agents. Recent advances in phage display technology allow isolation of high affinity recombinant antibody fragments (Fabs) that may bind unique epitopes of biological threat agents. The versatility of the selection process lends itself to efficient screening methodologies and can increase the number of antigen binding clones that can be isolated. Pilot scale biomanufacturing can then be used for the economical production of these immunoglobulin reagents in bacterial fermentation systems, and expression vectors with hexahistidine tags can be used to simplify downstream purification. One such Fab reagent directed against botulinum neurotoxin A/B has been shown to be sensitive in a variety of assay formats including surface plasmon resonance (SPR), flow cytometry, enzyme linked immunosorbent assay (ELISA), and hand-held immunochromatographic assay. Recombinant antibodies can provide another source of high quality detection reagents in our arsenal to identify or detect pathogens in environmental samples. 相似文献
7.
G L Rowley K E Rubenstein J Huisjen E F Ullman 《The Journal of biological chemistry》1975,250(10):3759-3766
Antimorphine antibodies inhibit the activity of morphine conjugates of mitochondrial malate dehydrogenase. Conjugation of malate dehydrogenase through tyrosine and amino groups resulted in only moderate losses of enzyme activity. On conjugation through disulfide bonds the enzyme activity first increased but dropped sharply with increasing substitution. Only the former conjugates were inhibited by excess antibodies. The degree of inhibition (up to 86%) was directly related to the number of morphine residues bonded directly to amino groups. The maximum number of antibody binding sites that bind to enzyme was nearly equal to the number of haptens provided there were 16 or less haptens/enzyme. However up to 26 haptens/enzyme became completely bound by antibody on long incubation. Inhibition of enzyme activity was detectably reduced by 2 times 10 minus 9 M morphine or 2 times 10 minus 10 M codeine, thus providing a sensitive assay for these drugs. The data suggest that enzyme inhibition occurs by conformational freezing of the enzyme when antibody binds to a morphine residue attached to one specific amino group. 相似文献
8.
A solid-phase immunosorbent assay for the detection of protein kinase-specific monoclonal antibodies
We describe two solid-phase immunosorbent assays that detect monoclonal antibodies against a cyclic AMP-dependent protein kinase from Paramecium tetraurelia without the need for pure kinase preparations. A radiometric immunosorbent assay (RISA) previously described by E.A. Pierce, M. C. Dame, and H. F. De Luca (1986, Anal. Biochem. 153, 67-74) was adapted to detect monoclonal antibodies against cyclic AMP-binding proteins. The RISA identified antibodies against the regulatory subunit of the enzyme, but failed to detect antibodies against the catalytic subunit. We therefore developed a solid-phase assay for immunoadsorbed protein kinase activity (IPKA) in 96-well plates. Antibodies were adsorbed from hybridoma supernatants to wells coated with anti-immunoglobulin antibodies. The wells were then incubated with protein kinase, and bound protein kinase activity was assayed with histone as a substrate. Monoclonal antibody concentrations of 1 micrograms/ml were reliably detected in hybridoma supernatants. As little as 10 histone-phosphorylating units (picomoles of phosphate incorporated per minute) were required per assay. The IPKA detected not only catalytic subunit-specific antibodies, but also antibodies directed against the regulatory subunit of the cyclic AMP-dependent protein kinase. Since crude preparations of protein kinase can be used in the IPKA, monoclonal antibodies raised against impure protein kinase can be identified. 相似文献
9.
《Journal of enzyme inhibition and medicinal chemistry》2013,28(6):955-960
AbstractCarbonic anhydrase 9 (CA9) and carbonic anhydrase 12 (CA12) were proposed as potential targets for cancer therapy more than 20 years ago. However, to date, there are only very few antibodies that have been described to specifically target CA9 and CA12 and also block the enzymatic activity of their targets. One of the early stage bottlenecks in identifying CA9- and CA12-inhibiting antibodies has been the lack of a high-throughput screening system that would allow for rapid assessment of inhibition of the targeted carbon dioxide hydratase activity of carbonic anhydrases. In this study, we show that measuring the esterase activity of carbonic anhydrase offers a robust and inexpensive screening method for identifying antibody candidates that block both hydratase and esterase activities of carbonic anhydrase’s. To our knowledge, this is the first implementation of a facile surrogate-screening assay to identify potential therapeutic antibodies that block the clinically relevant hydratase activity of carbonic anhydrases. 相似文献
10.
11.
Immunological comparisons of nitrate reductase of different plant species using monoclonal antibodies 总被引:2,自引:2,他引:0
Six monoclonal antibodies against different epitopes of maize leaf nitrate reductase were used to compare plant nitrate reductases in enzyme linked immunosorbent assay and enzyme activity inhibition tests. The number of cross-reacting antibodies was shown to vary with species according to phylogenetic classification, ranging from five (sugarcane) to one (dicotyledonous species). Cross-reactions were restricted to higher plant nitrate reductases. 相似文献
12.
Eleven hybridoma clones which secrete monoclonal antibodies against purified rat liver alcohol dehydrogenase (EC 1.1.1.1) were isolated. Antibodies (R-1-R-11) were identified by their ability to bind to immobilized pure alcohol dehydrogenase in an enzyme-linked immunoadsorbent assay, in which antibody R-9 showed the highest binding capacity. Except for R-1 and R-7, all antibodies inhibited catalytic activity of the enzyme isolated from inbred (Fischer-344) or outbred (Sprague-Dawley) strains (R-11 greater than R-9 greater than R-4 greater than R-6 greater than R-10 greater than R-8 greater than R-2 = R-3 = R-5). The inhibition of enzyme activity by antibodies was noncompetitive for ethanol and NAD+, and was dependent on antibody concentration and incubation time. Antibodies R-4, R-9, and R-11 were most effective when enzyme activity was assayed below pH 7.7-7.8, a condition thought to protonate the enzyme's active center. These three antibodies did not inhibit horse liver alcohol dehydrogenase activity, indicating their species specificity. Such antibodies will be useful to delineate structural and functional roles of rat liver alcohol dehydrogenase. 相似文献
13.
Shigeki Kimura Tomohiko Hayano Kanefusa Kato 《Biochimica et Biophysica Acta (BBA)/General Subjects》1984,799(3):252-259
Two monoclonal antibodies to human and bovine neuron-specific γγ enolase have been produced in the isolated hybrid cell lines, which were obtained by fusion between γγ-immunized mouse spleen cells and mouse myeloma cells (P3-NS-1/1-Ag4-1), followed by a screening procedure with an enzyme immunoassay. The monoclonal antibody to human γγ enolase (E1-G3) and that to bovine γγ enolase (B1-D6) consisted of γ2a/κ and γl/κ immunoglobulin chains, respectively. Both antibodies could bind with the respective antigen with a molar ratio of about 1:1, and were found to be specific for the γ subunit of enolase, showing reactivities with human γγ and αγ, rat γγ and αγ, and bovine γγ enolases. However, the antibodies did not cross-react with the α or β subunit of human and rat enolase isozymes. Both antibodies could partially inhibit the activity of γγ and αγ enolases. E1-G3 antibody inhibited γγ and αγ enolase activity by 70 and 30%, respectively, and B1-D6 antibody, by 90 and 40%, respectively. Both antibodies had no effect on the activity of αα and ββ enolases of human and rat origins. The applicability of E1-G3 and B1-D6 antibodies to the sandwich-type enzyme immunoassay for neuron-specific enolase (enolase γ subunit) was examined, and it was found that the assay system using E1-G3 and B1-D6 as the labeled antibodies were sufficiently sensitive for the assay of serum neuron-specific enolase concentrations. 相似文献
14.
V B Gervazieva I G Ovsiannikova N I Voronkin N N Sorochinskaia B N Ra?kis 《Zhurnal mikrobiologii, epidemiologii, i immunobiologii》1987,(9):33-35
An ELISA system for the detection of allergen-specific IgE antibodies to ragweed allergen has been developed. The system is highly sensitive and specific. Ragweed pollen allergen has been obtained by the dialysis of water-soluble extract through a kidney membrane. The high molecular fraction of ragweed allergen, showing the whole of the allergenic activity detected by skin tests in untreated patients, has been used for coating polystyrene assay plates. To detect IgE antibodies to ragweed allergen, the conjugate of sheep anti-IgE antibodies with horse-radish peroxidase has been used. The level of allergen-specific IgE antibodies has been determined on the basis of the data on the optical density of the samples in comparison with that of the normal sera. The correlation factor of the results obtained in the assay of specific IgE antibodies with the newly developed assay system and with the commercial kit Phadezyme RAST manufactured by Pharmacia AB (Sweden) has proved to be 0.82 at n = 39, p less than 0.01, while the variation factor in the reproduction of the assay results has proved to be 12% at n = 40. 相似文献
15.
Production, characterization, and applications of monoclonal antibodies reactive with soybean nodule xanthine dehydrogenase 总被引:1,自引:1,他引:0
Seven monoclonal antibodies were produced against soybean nodule xanthine dehydrogenase, an enzyme involved in ureide synthesis. Specificity of the seven monoclonal antibodies for xanthine dehydrogenase was demonstrated by immunopurifying the enzyme to homogeneity from a crude nodule extract using antibodies immobilized to Sepharose 4B beads. Each monoclonal antibody was covalently bound to Sepharose 4B beads for the preparation of immunoaffinity columns for each antibody. All seven antibodies were found to be of the IgG1,K subclass. A competitive, indirect enzyme-linked immunosorbent assay demonstrated that two of the seven antibodies shared a common epitope while the remaining five antibodies defined unique determinants on the protein. Rapid, large scale purification of active xanthine dehydrogenase to homogeneity was performed by immunoaffinity chromatography. The presence of xanthine dehydrogenase activity and protein in every organ of the soybean plant was determined. Crude extracts of nodules, roots, stems, and leaves cross-reacted with all seven monoclonal antibodies in an indirect enzyme-linked immunosorbent assay. A positive correlation was observed between the degree of cross-reactivity of a given organ and the level of enzyme activity in that organ. These data demonstrate that xanthine dehydrogenase is not nodule specific. Antigenic variability of xanthine dehydrogenase present in crude extracts from nodules of soybean, wild soybean, cowpea, lima bean, pea, and lupin were detected in the indirect enzyme-linked immunosorbent assay which corresponded to six binding patterns for xanthine dehydrogenase from these plant species. These results correspond well with the epitope determination data which showed that the seven antibodies bind to six different binding determinants on the enzyme. 相似文献
16.
Functional heterogeneity of monoclonal antibodies obtained using different screening assays 总被引:10,自引:0,他引:10
Two alternate screening methods have enabled the detection of monoclonal antibodies with different specificities toward the lysosomal enzyme alpha-mannosidase of Dictyostelium discoideum. Spleen/myeloma hybrid cell cultures were screened for antibody production by separate assays: an indirect enzyme-linked immunoadsorbent assay (ELISA) based on the antibody binding to enzyme adsorbed on plastic, and a direct assay of the antibodies' ability to precipitate enzyme activity with fixed Staphylococcus aureus cells (Pansorbin). Fourteen stable antibody-producing cell lines resulted from a single fusion; these fell into three distinct classes based on their screening characteristics. A group of eight were positive in both assays, and these immunoprecipitated a 140,000 Mr precursor form of alpha-mannosidase in addition to the 58,000 and 60,000 Mr mature enzyme subunits from [35S]methionine-labeled total secreted protein preparations. Two of the antibodies were positive only in the immunoprecipitation assay; these failed to precipitate the 140,000 Mr precursor. The third class consisted of four antibodies that were positive only in the ELISA method. These exclusively recognized an altered conformation of the enzyme (precursor and mature forms) that was immobilized either on plastic or on nitrocellulose paper. In addition, only members of this class were able to bind to immobilized fragments of protease-treated enzyme. The implications of these findings for the general design of monoclonal antibody screenings and for the alternative structures of this enzyme are discussed. 相似文献
17.
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
18.
The use of a nitrocellulose-enzyme immunoassay for the rapid screening of monoclonal antibodies to human enteroviruses 总被引:2,自引:0,他引:2
A simple, indirect enzyme immunoassay using purified enterovirus adsorbed on to nitrocellulose has been developed for screening monoclonal antibodies to enteroviruses. The sensitivity of the assay ranged from 10 ng to 1 microgram of viral protein and was 10- to 50-fold more sensitive than conventional EIA on microplates. This simple, sensitive and specific assay proved to be a useful and practical tool for detecting monoclonal antibodies which would not be found in a conventional EIA screening procedure. 相似文献
19.
A A Sokolenko M N Slavin K L Shakhanina 《Zhurnal mikrobiologii, epidemiologii, i immunobiologii》1988,(2):99-103
A scheme of the purification of hepatitis B virus surface antigen (HBsAg) as applied to the enzyme immunoassay (EIA) for the detection of antibodies to HBsAg is described. An indirect EIA technique for the detection of IgG and IgM antibodies to HBsAg has been developed and the diagnostic assay system based on the use of immunoreagents and solid-phase carriers produced in the USSR has been obtained. The sensitivity of the indirect EIA technique in the detection of IgG antibodies to HBsAg exceeds that of double immunodiffusion in gel used for this purpose 2,500- to 5,000-fold. The study has shown the possibility of using the indirect EIA technique for the detection of antibodies to HBsAg, both free and bound in immune complexes, of detecting antibodies to HBsAg in patients with acute and chronic viral hepatitis B, as well as of simultaneous detection of IgG and IgM antibodies to HBsAg without pseudonegative results. 相似文献
20.
Monoclonal hybridoma antibodies directed against RNA polymerase from E. coli have been obtained. Only a few have been found to inhibit the enzyme activity. Antibodies produced by two clones, PYN-1 and PYN-2, inhibit RNA polymerase at the stage of RNA chain elongation. The PYN-1 antibodies react with the beta'-subunit of the enzyme. The PYN-2 antibodies react with the beta-subunit and with its 130 kDa amber fragment. 相似文献