Academic literature on the topic 'Polyclonal antiserum AS'
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Journal articles on the topic "Polyclonal antiserum AS"
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Köves, K., and A. Arimura. "Solid-phase adsorption method for removing undesired antibodies from polyclonal antiserum." Journal of Histochemistry & Cytochemistry 37, no. 6 (1989): 903–8. http://dx.doi.org/10.1177/37.6.2723405.
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We have developed a novel and simple method, requiring only a small amount of antigen, for removal of undesired antibodies from antiserum. The method was established using a well-characterized antiserum against rat luteinizing hormone (anti-rLH). Wells of polystyrene tissue culture plates were coated with rat LH (rLH). Anti-rLH diluted 1:3000 was added to rLH-coated wells and shaken to remove LH antibodies. Control anti-rLH was treated in a similar manner in non-rLH-coated wells. Both antisera were tested by immunocytochemistry on rat pituitaries. Antiserum from rLH-coated wells stained no cells, whereas the control serum stained cells that were morphologically typical of LH cells. The effectiveness of this antibody removal was also confirmed in a modified ELISA. In another experiment, anti-rLH and anti-hTSH beta sera were mixed. The final dilution of both antisera was 1:10,000. Anti-rLH was removed by the purification method described. Completeness of antibody removal was confirmed by a double-immunohistochemical staining of rat pituitary in which sections were first stained by the PAP method and then stained with an immunofluorescence procedure after elution of the first antigen-antibody complex. The mixed antiserum incubated in rLH-coated wells did not stain LH cells. There was no co-localization between the LH immunopositivity demonstrated by an anti-rLH serum using immunofluorescence and cells immunostained with the purified antiserum using the PAP method. As indicated in ELISA, the titer of the TSH beta antiserum was not decreased compared to that of the untreated, mixed control antiserum, and the LH antibodies were eliminated by the treatment. This new purification method has four distinct advantages: (a) antiserum is not treated chemically; (b) it requires only a small amount of antigen compared with the amount required for affinity chromatography; (c) neither the undesired antigen-antibody complex(es) nor an excess amount of antigen is present in the purified antiserum; and (d) removal of undesired antibodies can be monitored by ELISA.
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Hoving, H. J. T., J. D. Venter, D. E. Worst, and M. R. Lipinski. "Adaptation of an immunodot assay for multiple prey identification of squid paralarvae in field trials." Journal of the Marine Biological Association of the United Kingdom 85, no. 6 (2005): 1499–501. http://dx.doi.org/10.1017/s0025315405012695.
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An optimized method, using polyclonal antibodies in an immunoassay, for prey detection in the diet of paralarvae of South African Loligo reynaudii is described. The study has increased the specificity of the antisera by determining the optimum antiserum dilutions and the detection limits of the antisera. Unfed laboratory-hatched paralarvae (negative control) were exposed to antisera and showed cross-reactions with polychaete antiserum.
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Shahangian, S., K. A. Agee, and R. P. Dickinson. "Concentration Dependencies of Immunoturbidimetric Dose-Response Curves: Immunoturbidimetric Titer and Reactivity, and Relevance to Design of Turbidimetric Immunoassays." Clinical Chemistry 38, no. 6 (1992): 831–40. http://dx.doi.org/10.1093/clinchem/38.6.831.
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Abstract To characterize polyclonal antisera for two-point immunoturbidimetric applications, we defined, as functions of antiserum concentration, two parameters derived from dose-response curves: the maximum bichromatic optical response, Tmax, and the antigen concentration in the region of excess antibody corresponding to one-half Tmax, or C50. We raised monospecific polyclonal antisera in goats against several human immunoglobulins, C-reactive protein, C3, C4, apolipoproteins A-I and B, and several other proteins. We could linearly relate the logarithm of the antiserum concentration to log C50 and to log Tmax. The concentration of polyethylene glycol affected not only C50 and Tmax but also their functional dependencies on antiserum concentration. We devised two definitions of immunoturbidimetric titer and related them to the titer obtained by the single radial immunodiffusion method of Becker (Immunochemistry 1969; 6:539-46).
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Parry, R. A., C. B. McLean, M. R. Alderton, P. J. Coloe, and A. C. Lawrie. "Polyclonal antisera to epacrid mycorrhizae and to Hymenoscyphus ericae display specificity." Canadian Journal of Botany 78, no. 7 (2000): 841–50. http://dx.doi.org/10.1139/b00-052.
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Three polyclonal antisera produced in mice were used to investigate specificity and cross-reactivity between ericaceous and epacridaceous mycorrhizal fungi. One antiserum was to a culture of Hymenoscyphus ericae (Read) Korf and Kernan, the fungal endophyte of Calluna vulgaris (L.) Hull (Ericaceae). The other two were to peloton preparations from roots of Epacris impressa Labill. (Epacridaceae) from two sites (Cranbourne and Grampians) in Victoria, Australia. By immunofluorescence, all three antisera recognised H. ericae but not Oidiodendron griseum Roback, suggesting a serological relationship with the former endophyte. They also recognised 10 of the 12 fungal isolates tested, from mycorrhizal roots of E. impressa (Cranbourne), and all 4 isolates from Astroloma pinifolium (R. Br.) Benth. (Epacridaceae) (Grampians). Furthermore, none of the antisera recognised any of the nine common soil-inhabiting fungi selected for screening. Antisera recognised only unmelanized hyphae on epacrid and other plant roots taken from the wild. With plants from Cranbourne, all antisera except the Grampians antiserum recognised hyphae only on epacrid roots, demonstrating specificity. Hyphae on other plant roots were not recognised by any of the antisera. With plants from the Grampians, all antisera recognised some hyphae on both epacrid and other plant roots, except in two instances. The immunogold labelling indicates that the antisera are specific for fungi and do not recognise the plant. Since the fungal isolate forms true mycorrhizal structures, this suggests that there is a serological similarity between fungi forming epacrid mycorrhiza and those (H. ericae) forming ericoid mycorrhiza.Key words: ericoid mycorrhizae, Epacridaceae, polyclonal antibodies, immunofluorescence, immunogold.
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CLEVELAND, S. M., H. P. TAYLOR, and N. J. DIMMOCK. "Selection of neutralizing antibody escape mutants with type A influenza virus HA-specific polyclonal antisera: possible significance for antigenic drift." Epidemiology and Infection 118, no. 2 (1997): 149–54. http://dx.doi.org/10.1017/s0950268896007303.
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Ten antisera were produced in rabbits by two or three intravenous injections of inactivated whole influenza type A virions. All contained haemagglutination-inhibition (HI) antibody directed predominantly to an epitope in antigenic site B and, in addition, various amounts of antibodies to an epitope in site A and in site D. The ability of untreated antisera to select neutralization escape mutants was investigated by incubating virus possessing the homologous haemagglutinin with antiserum adjusted to contain anti-B epitope HI titres of 100, 1000 and 10000 HIU/ml. Virus-antiserum mixtures were inoculated into embryonated hen's eggs, and progeny virus examined without further selection. Forty percent of the antisera at a titre of 1000 HIU/ml selected neutralizing antibody escape mutants as defined by their lack of reactivity to Mab HC10 (site B), and unchanged reactivity to other Mabs to site A and site D epitopes. All escape mutant-selecting antisera had a ratio of anti-site B (HC10)-epitope antibody[ratio ]other antibodies of [ges ]2·0[ratio ]1. The antiserum with the highest ratio (7·4[ratio ]1) selected escape mutants in all eggs tested in four different experiments. No antiserum used at a titre of 10000 HIU/ml allowed multiplication of any virus. All antisera used at a titre of 100 HIU/ml permitted virus growth, but this was wild-type (wt) virus. We conclude that a predominant epitope-specific antibody response, a titre of [ges ]1000 HIU/ml, and a low absolute titre of other antibodies ([les ]500 HIU/ml) are three requirements for the selection of escape mutants. None of the antisera in this study could have selected escape mutants without an appropriate dilution factor, so the occurrence of an escape mutant-selecting antiserum in nature is likely to be a rare event.
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Michon, Francis, Samuel L. Moore, John Kim, et al. "Doubly Branched Hexasaccharide Epitope on the Cell Wall Polysaccharide of Group A Streptococci Recognized by Human and Rabbit Antisera." Infection and Immunity 73, no. 10 (2005): 6383–89. http://dx.doi.org/10.1128/iai.73.10.6383-6389.2005.
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ABSTRACT A number of epitope specificities associated with the cell wall polysaccharide antigen of group A streptococci were identified in a polyclonal rabbit antiserum induced in rabbits by whole group A streptococci and in polyclonal convalescent human antisera from children that had recovered from streptococcal A infections. The identification was achieved by using a series of synthetic oligosaccharides, glycoconjugates, and bacterial polysaccharide inhibitors to inhibit the binding of the group A helical polysaccharide to the polyclonal antisera. The exclusively dominant epitope expressed in the convalescent human antisera was the doubly branched extended helical hexasaccharide with the structure α-l-Rhap(1→2)[β-d-GlcpNAc(1→3)]α-l-Rhap(1→3)α-l-Rhap(1→2)[β-d-GlcpNAc(1→3)]α-l-Rhap. The hexasaccharide epitope also bound with the highest immunoreactivity to the rabbit antiserum. In contrast, the human antisera did not show significant binding to the singly branched pentasaccharide with the structure α-l-Rhap(1→2)α-l-Rhap(1→3)α-l-Rhap(1→2)[β-d-GlcpNAc(1→3)]α-l-Rhap or the branched trisaccharide α-l-Rhap(1→2)[β-d-GlcpNAc(1→3)]α-l-Rhap, although both these haptens bound significantly to the same rabbit antiserum, albeit with less immunoreactivity than the hexasaccharide. Inhibition studies using streptococcal group A and B rabbit antisera and the inhibitors indicated above also suggested that the group A carbohydrate, unlike the group B streptococcal polysaccharide, does not contain the disaccharide α-l-Rhap(1→2)α-l-Rhap motif at its nonreducing chain terminus, stressing the importance of mapping the determinant specificities of these two important streptococcal subcapsular group polysaccharides to fully understand the serological relationships between group A and group B streptococci.
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Wassef, Nabila M., Glenn M. Swartz Jr., Carl R. Alving, and Morris Kates. "Antibodies to liposomal phosphatidylcholine and phosphatidylsulfocholine." Biochemistry and Cell Biology 68, no. 1 (1990): 54–64. http://dx.doi.org/10.1139/o90-007.
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Antibodies against dimyristoyl phosphatidylsulfocholine or dimyristoyl phosphatidylcholine were raised in rabbits after injection of liposomes containing phosphatidylsulfocholine or phosphatidylcholine, cholesterol, and lipid A. The antibody activities were assayed by complement-dependent immune damage to liposomes and by a solid-phase, enzyme-linked immunosorbent assay using purified dimyristoyl phosphatidylcholine or dimyristoyl phosphatidylsulfocholine as antigen. Each antiserum raised against phosphatidylsulfocholine reacted with liposomes containing phosphatidylcholine, and each antiserum raised against phosphatidylcholine reacted with liposomes containing phosphatidylsulfocholine. However, adsorption of dimyristoyl phosphatidylsulfocholine antiserum with liposomes containing dimyristoyl phosphatidylcholine removed all activity against dimyristoyl phosphatidylcholine, but did not eliminate antibody activity against dimyristoyl phosphatidylsulfocholine. These results indicate that the antiserum against phosphatidylsulfocholine contained mixed populations of antibodies. Polyclonal antisera that have been appropriately adsorbed can therefore be obtained with a high degree of specificity for phosphatidylsulfocholine and such antisera can distinguish between phosphatidylsulfocholine and phosphatidylcholine.Key words: liposomes, antibodies, phosphatidylsulfocholine, phosphatidylcholine.
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Abouzid, A. M., J. Freitas-Astua, D. E. Purcifull, et al. "Serological Studies Using Polyclonal Antisera Prepared Against the Viral Coat Protein of Four Begomoviruses Expressed in Escherichia coli." Plant Disease 86, no. 10 (2002): 1109–14. http://dx.doi.org/10.1094/pdis.2002.86.10.1109.
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Polyclonal rabbit antisera were produced to the coat protein of Bean golden mosaic virus Brazil isolate (BGMV), Cabbage leaf curl virus (CabLCV), Tomato yellow leaf curl virus (TYLCV), and Tomato mottle virus (ToMoV), all expressed in Escherichia coli by the pETh expression vector. The expressed coat protein of each virus was purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis for use as an immunogen. The antisera to BGMV, CabLCV, TYLCV, and ToMoV reacted in indirect (plate-trapping) enzyme-linked immunosorbent assay (ELISA) with extracts from begomovirus-infected tissue. The antisera to BGMV, CabLCV, TYLCV, and ToMoV also reacted specifically with the test begomovirus antigens in leaf imprint blots and Western blots. The CabLCV and TYLCV antisera were used to detect Bean golden yellow mosaic virus antigens by immunogold labeling of thin sections of infected bean tissues. In tissue blot immunoassays, the TYLCV antiserum reacted well with TYLCV antigens but not with ToMoV antigens, while CabLCV antiserum reacted well with ToMoV antigens and weakly with TYLCV antigens. The results indicate that polyclonal antisera prepared to expressed begomovirus coat proteins were useful for the detection of begomoviruses in an array of assays.
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Banerji, Benoy, and Carl R. Alving. "Antibodies to liposomal phosphatidylserine and phosphatidic acid." Biochemistry and Cell Biology 68, no. 1 (1990): 96–101. http://dx.doi.org/10.1139/o90-012.
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Polyclonal antisera to phosphatidylserine or phosphatidic acid were induced in rabbits by injecting liposomes containing phosphatidylserine or phosphatidic acid and lipid A. Adsorption of antisera with liposomes containing different phospholipids revealed that some degree of reactivity with one or more phospholipids other than the immunizing phospholipid was often observed. However, cross-reactivity with other phospholipids was not a universal phenomenon, and one antiserum to phosphatidylserine failed to cross-react (i.e., was not adsorbed) with liposomes containing other phospholipids. All of the antisera were inhibited by soluble phosphorylated haptens (e.g., phosphocholine but not choline), but one of the antisera to phosphatidylserine was inhibited both by phosphoserine and by serine alone. Liposomal membrane composition influenced the activity of antiserum to phosphatidylserine. Regardless of whether unsaturated (beef brain) or saturated (dimyristoyl) phosphatidylserine was used in the immunizing liposomes, the antisera reacted more vigorously with liposomes containing unsaturated than saturated phosphatidylserine. We conclude that liposomes containing lipid A can serve as vehicles for stimulating polyclonal antisera to phosphatidylserine and phosphatidic acid. Although cross-reactivity with certain other phospholipids can be observed, sera from selected animals apparently can exhibit a high degree of specific activity to the immunizing phospholipid antigen.Key words: liposomes, antibodies, phospholipids, phosphatidylserine, phosphatidic acid.
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Bartlett, Marilyn S., William C. Angus, Margaret M. Shaw, et al. "Antibody to Pneumocystis cariniiProtects Rats and Mice from Developing Pneumonia." Clinical Diagnostic Laboratory Immunology 5, no. 1 (1998): 74–77. http://dx.doi.org/10.1128/cdli.5.1.74-77.1998.
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ABSTRACT Well-proven mouse and rat models were used to show that polyclonal antisera to Pneumocystis carinii protect against P. carinii pneumonia. Antibodies were obtained from animals that were allowed to recover from severe P. carinii pneumonia after immunosuppression had been stopped and which then were given a booster injection of P. carinii from the same animal species. Mice immunosuppressed with corticosteroids or antibodies to L3T4+ lymphocytes (which are comparable to CD4 cells of humans) and transtracheally inoculated with mouse P. carinii did not develop P. carinii pneumonia if they were passively immunized with antiserum, while mice immunosuppressed and inoculated by identical procedures but not given antibodies developed severe infections. Rats immunosuppressed with corticosteroids and inoculated with rat P. carinii had less severe infections if they were given rat anti-P. carinii antisera. The polyclonal antisera developed in mice provided greater protection for the mice than the polyclonal rat antisera did for the rats; however, the potencies and compositions of the antisera were not quantitated and probably differed. Since both rats and mice can be protected from P. carinii infections with polyclonal antisera, it may be possible to develop vaccines that will elicit protective antibodies in humans.
Dissertations / Theses on the topic "Polyclonal antiserum AS"
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Pongo, Elizabeth C. "The role of E2A proteins in pancreatic beta cells and the characterization of an anti-E2A specific polyclonal antiserum." Scholarly Commons, 1997. https://scholarlycommons.pacific.edu/uop_etds/2303.
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Basic helix-loop-helix (bHLH) proteins belong to a class of transcription factors that are critical regulators of development, cell growth and differentiation. One particular family member includes the products of the E2A gene, E12 (Pan-2) and E47 (Pan-1), ubiquitous transcription factors localized in the nucleus. E12 and E47 gene products are generated by alternative RNA splicing. E12 and E47 proteins have been implicated as transcriptional regulators of the rat I insulin gene, immunoglobulin light and heavy chain genes and several muscle-specific genes. To delineate the role ofE2A proteins in directing insulin gene transcription, we have characterized an anti-E2A polyclonal antiserum which recognizes both E12 and E47 and used this reagent to study E2A proteins in the pancreas. In these studies, we have demonstrated that the anti-E2A polyclonal antiserum is highly specific for E2A proteins in a variety of different cell lines representing different tissues. Furthermore, using this immunohistochemical tool, we have demonstrated that E2A proteins are posttranslationally modified in beta cells, insulin producing cells in the pancreas. We also provide evidence that a posttranslationally modified form of E2A protein is involved in glucose-induced insulin gene transcription in beta cells. Lastly, we provide evidence that E2A proteins are associated with other bHLH proteins or other non-bHLH proteins in pancreatic beta cells.
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Pereira, Ana Cecília Bergamim. "Proteína capsidial do Rupestris stem pitting-associated vírus : seqüenciamento do gene, expressão em Escherichia coli, purificação e produção de anti-soro policlonal /." São José do Rio Preto : [s.n.], 2008. http://hdl.handle.net/11449/94835.
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Orientador: José Osmar Gaspar<br>Banca: Hugo Kuniyuki<br>Banca: Fátima Pereira de Souza<br>Resumo: O lenho estriado de rupestris ou cascudo (Rupestris stem pitting - RSP), um dos componentes do Complexo do lenho rugoso ("Rugose wood" - RW), é considerado uma das doenças de videira transmitidas por enxertia de grande relevância econômica para a viticultura. O Rupestris stem pitting associated virus - RSPaV foi associado com a doença do lenho estriado ou cascudo, sendo classificado como espécie do gênero Foveavirus, pertencente a família Flexiviridae. No presente trabalho, descrevem-se o sequenciamento do gene da proteína capsidial (CP) de um isolado brasileiro do RSPaV (RSPaV-SP), sua expressão em Escherichia coli, purificação da proteína capsidial recombinante e a produção de anti-soro policlonal em coelho. O sequenciamento do gene resultou em uma seqüência de 780 nucleotídeos e 259 aminoácidos deduzidos com massa molecular estimada de 28 kDa. A análise filogenética, entre a seqüência correspondente à CP do RSPaV-SP e outras variantes do mesmo vírus, evidenciou a formação de 4 grupos distintos, sendo o isolado brasileiro incluído no grupo da variante BS do RSPaV. A proteína capsidial recombinante foi purificada em coluna de afinidade e apresentou massa molecular estimada de 32kDa (4kDa da seqüência do vetor e 28kD da CP do RSPaV-SP). O anti-soro produzido apresentou-se específico na detecção da proteína capsidial recombinante purificada por "Western-blot", sem reação com proteína heteróloga a partir da diluição 1:4000. Nesta diluição, o anti-soro foi efetivo na detecção do vírus em extratos de plantas infectadas, sendo que nenhuma reação foi observada com extratos de plantas sadias. Considerando-se que este vírus apresenta variações de concentração na planta durante as estações do ano, e que, os testes sorológicos foram realizados durante a estação de baixa concentração do vírus, os resultados ...(Resumo completo, clicar acesso eletrônico abaixo)<br>Abstract: Rupestris stem pitting (RSP), a component of the rugose wood (RW) complex, is one of the most graft-transmissible grapevine virus diseases with great economic importance for viticulture . Rupestris stem pitting-associated virus (RSPaV), genus Foveavirus within the family Flexiviridae, has been associated with this disease. This work reports the sequencing of the coat protein (CP) gene of a brazilian an isolate of RSPaV (RSPaV-SP), its expression in Escherichia coli, purification of the recombinant coat protein and production of a polyclonal antiserum in rabbit. CP gene was found to be 780nt long, with a 256 deduced amino acid sequence encoding a predicted protein of 28 kDa. In filogenetic analysis, with RSPaV-SP and other variants of the virus, four groups were found and the sequence of RSPaV-SP showed the highest identity with the variant RSPaV-BS. The recombinant coat protein was purified by affinity chromatography and showed a molecular weight of 32kDa (4 kDa from a small vector sequence plus 28 kDa for the CP of RSPaV-SP). The antiserum proved specific for detection of the recombinant protein by Western Blot, and did not react with heterologous proteins starting at a dilution of 1:4000. At this dilution, the antiserum was effective in the virus detection of leaf extracts of infected plants and no reaction was observed with extracts from healthy grapevines. Considering that the virus is found at low concentrations in the plants during the seasons of the year, the results obtained so far were highly satisfactory for RSPaV detection. Serological methods have advantages over the biological indexing method, since they are cheaper and can be used in large-scale tests such as ELISA. Experiments using the ELISA technique were not successful. Purification of the native recombinant protein would be an alternative more efective to detect the virus using these technique.<br>Mestre
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Liu, Catherine Heung Luen. "Expression of an EF-1a like rat cDNA, S1, in Escherichia coli and production of a rabbit polyclonal antiserum to the recombinant protein." Thesis, McGill University, 1992. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=61334.
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A previously identified rat cDNA (S1) that shares 78% nucleotide homology and a predicted 92% amino acid sequence homology with human EF-1$ alpha$ was used to express S1 in E. coli and to generate a polyclonal antibody to pS1. A recombinant plasmid pGEX-2T-S1 was constructed, containing the glutathione S-transferase gene. The expressed fusion protein was purified and digested with thrombin to produce a recombinant S1 protein (rpS1) containing slightly modified N-terminus. Purified rpS1 was used to raise a rabbit antiserum which recognized rpS1 on immunoblots. A polyclonal antiserum to EF-1$ alpha$ failed to react with rpS1. Similarly the anti-rpS1 does not recognize EF-1$ alpha$ on immunoblots. Anti-rpS1 therefore is able to distinguish pS1 from EF-1$ alpha$ despite their extensive amino acid sequence homology. Anti-rpS1 and anti-Ef-1$ alpha$ will be used to study the similarities and differences between pS1 and EF-1$ alpha$ in vivo and in vitro.
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Pinto, Luiz Rafael. "Tomato chlorosis virus: purificação, produção de antissoro, reação de genótipos e avaliação de danos em batateira." Universidade de São Paulo, 2018. http://www.teses.usp.br/teses/disponiveis/11/11135/tde-29062018-091055/.
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O Tomato chlorosis virus (ToCV) é uma espécie do gênero Crinivirus que causa danos, principalmente na cultura do tomateiro (Solanum lycopersicum). Foi primeiramente isolado e descrito em 1998, nos Estados Unidos, e em seguida foi reportado em doze países. No Brasil, foi constatado primeiramente no Estado de São Paulo, na região de Sumaré, em 2008, e posteriormente nos Estados da Bahia, Espírito Santo, Goiás, Minas Gerais e Rio de Janeiro. Há evidência da sua presença também nos Estados do Paraná e Santa Catarina. O ToCV pode infectar outras solanáceas além do tomateiro e, recentemente, foi observado infectando plantas de batata (Solanum tuberosum) no Brasil. Esse crinivirus é transmitido no Brasil principalmente pelo aleirodídeo (mosca branca) Bemisia tabaci MEAM1. Considerando o patossistema batateira/ToCV, não há estudos sobre a ocorrência, sintomatologia em diferentes variedades e danos provocados por esse crinivirus. Também não há antissoro policlonal para o isolado brasileiro do ToCV para uso na diagnose da doença em solanáceas. Esse trabalho teve por objetivos: purificar o ToCV e produzir antissoro policlonal; avaliar a reação de genótipos de batateira à infecção com o ToCV; avaliar o dano provocado por esse vírus em duas variedades de batateira. A purificação do vírus a partir de folhas de tomateiro e a produção de antissoro policlonal em coelho foram satisfatórias. No entanto, o antissoro não foi eficiente em ELISA, mas sim em dot-blot e somente na diluição de 1:20. Foi avaliada a reação de 21 genótipos de batateira à infecção com o ToCV, por meio da inoculação com B. tabaci MEAM1, com chance de escolha do vetor. Nenhum genótipo exibiu resistência à infecção; enquanto a variedade Camila foi assintomática e não apresentou alteração na fotossíntese. Plantas de batateira das variedades Ágata e Asterix sadias foram inoculadas com o ToCV, por meio da B. tabaci MEAM1 e ao final foram avaliadas a massa fresca da parte aérea, peso e número dos tubérculos colhidos. Em dois experimentos independentes, as reduções médias no peso fresco da parte aérea foram de 60,1% para Ágata e 46% para Asterix. Porém, as reduções nas produções dessas variedades, no primeiro experimento foram de 99,5% e 98,1%, respectivamente; enquanto no segundo os valores foram de 82,3% e 56,2%, respectivamente.<br>Tomato chlorosis virus (ToCV) is a species of the genus Crinivirus, which is causing considerable losses mainly on tomato crop (Solanum lycopersicum). It was first isolated and described on 1998 in the United States and subsequently reported in twelve countries. In Brazil, it was first reported in São Paulo State, in Sumaré region in 2008, and after that on the states of Bahia, Espírito Santo, Goiás, Minas Gerais and Rio de Janeiro. There is evidence of the presence of ToCV on the states of Paraná and Santa Catarina. ToCV can also infect other solanaceae and more recently, it was reported infecting potato plants (Solanum tuberosum) in Brazil. This crinivirus is transmitted by Bemisia tabaci MEAM1. Considering the patosystem potato/ToCV, there are no studies on the occurrence, symptomatology in different varieties, and damages caused by this crinivirus. In addition, there is no polyclonal antiserum for the Brazilian isolate of ToCV for use in diagnosis. The objectives of the present work were: to purify the virus and produce a polyclonal antiserum; to evaluate the reaction of potato genotypes to ToCV infection; to evaluate the yield loss caused by this crinivirus on two potato cultivars. The virus purification from tomato leaves and the production of polyclonal antiserum in rabbit were satisfactorily accomplished. However, the antiserum was not efficient on ELISA test, but in dot-blot, only when diluted 1:20. The reaction of 21 potato genotypes to infection with ToCV was evaluated by inoculation with B. tabaci MEAM1, with chance of choice for the vector. All genotypes were infected with ToCV and Camila was the only one asymptomatic. Plants of cultivars Ágata and Asterix were inoculated with ToCV, by means of viruliferous vector, and at the end were evaluated for the fresh mass of the aerial part, weight and number of harvested tubers. In two independent experiments, average reductions in aerial fresh weight were 60.1% for Ágata and 46% for Asterix. However, reductions in yield of these varieties in the first experiment were 99.5% and 98.1%, respectively; while in the second the values were 82.3% and 56.2%, respectively.
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La, Mattina Coby Ann. "Identification of an aqueous glue protein, SCP-2, and the development of a polyclonal antiserum against the bHLH transcription factor SGSF in Latrodectus Hesperus." Scholarly Commons, 2009. https://scholarlycommons.pacific.edu/uop_etds/716.
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Although numerous spider fibroins have been reported, no known silk coating peptides have been discovered. We provide the first biochemical evidence for a spider coating peptide, called SCP-2, found on gumfooted lines, scaffolding joints and egg cases. The presence of this spider coating peptide on the fibers is supported by MS/MS analysis. Using quantitative real-time PCR analysis, we also demonstrate that SCP-2 has a flagelliform-restricted mRNA pattern of expression. Molecular modeling of the SCP-2 amino acid sequence predicts it adopts an alpha-helical structure that is amphipathic in nature. SCP-2, which can be extracted from fibers using water, is hypothesized to influence the mechanical properties of the silk fibers as well as serve a protective function for the threads. Based upon the restricted pattern of expression of SCP-2, our findings reveal novel insight regarding the glandular function of the flagelliform gland in . cob weaving spiders, suggesting it produces aqueous coating materials that are deposited on a wide range of different silk types. In addition, in an attempt to advance our understanding regarding silk gene transcription, our lab has developed the first antibody against the bHLH factor SGSF. SGSF has been implicated as a potential transcriptional regulator of silk gene transcription in spiders. Development of the anti-SGSF antibody was accomplished via the overexpression and purification of a fusion protein in bacteria, which consisted of the C-terminal region of SGSF fused to thioredoxin. Purified SGSF fusion proteins were injected into rabbits and the polyclonal antiserum was collected and tested by western blot analysis to determine the specificity of the immunological reagent. Western blot analyses revealed the anti-SGSF antiserum was capable of recognizing bacterially expressed SGSF in an efficient manner. Collectively, these studies lay the groundwork for future investigations involving the use of the antibody to determine the role of SGSF in silk transcription.
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Pereira, Ana Cecília Bergamim [UNESP]. "Proteína capsidial do Rupestris stem pitting-associated vírus: seqüenciamento do gene, expressão em Escherichia coli, purificação e produção de anti-soro policlonal." Universidade Estadual Paulista (UNESP), 2008. http://hdl.handle.net/11449/94835.
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pereira_acb_me_sjrp.pdf: 828257 bytes, checksum: de1b44b38dfac1b95be8ef5a683e7543 (MD5)<br>Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)<br>O lenho estriado de rupestris ou cascudo (Rupestris stem pitting – RSP), um dos componentes do Complexo do lenho rugoso (“Rugose wood” - RW), é considerado uma das doenças de videira transmitidas por enxertia de grande relevância econômica para a viticultura. O Rupestris stem pitting associated virus – RSPaV foi associado com a doença do lenho estriado ou cascudo, sendo classificado como espécie do gênero Foveavirus, pertencente a família Flexiviridae. No presente trabalho, descrevem-se o sequenciamento do gene da proteína capsidial (CP) de um isolado brasileiro do RSPaV (RSPaV-SP), sua expressão em Escherichia coli, purificação da proteína capsidial recombinante e a produção de anti-soro policlonal em coelho. O sequenciamento do gene resultou em uma seqüência de 780 nucleotídeos e 259 aminoácidos deduzidos com massa molecular estimada de 28 kDa. A análise filogenética, entre a seqüência correspondente à CP do RSPaV-SP e outras variantes do mesmo vírus, evidenciou a formação de 4 grupos distintos, sendo o isolado brasileiro incluído no grupo da variante BS do RSPaV. A proteína capsidial recombinante foi purificada em coluna de afinidade e apresentou massa molecular estimada de 32kDa (4kDa da seqüência do vetor e 28kD da CP do RSPaV-SP). O anti-soro produzido apresentou-se específico na detecção da proteína capsidial recombinante purificada por “Western-blot”, sem reação com proteína heteróloga a partir da diluição 1:4000. Nesta diluição, o anti-soro foi efetivo na detecção do vírus em extratos de plantas infectadas, sendo que nenhuma reação foi observada com extratos de plantas sadias. Considerando-se que este vírus apresenta variações de concentração na planta durante as estações do ano, e que, os testes sorológicos foram realizados durante a estação de baixa concentração do vírus, os resultados...<br>Rupestris stem pitting (RSP), a component of the rugose wood (RW) complex, is one of the most graft-transmissible grapevine virus diseases with great economic importance for viticulture . Rupestris stem pitting-associated virus (RSPaV), genus Foveavirus within the family Flexiviridae, has been associated with this disease. This work reports the sequencing of the coat protein (CP) gene of a brazilian an isolate of RSPaV (RSPaV-SP), its expression in Escherichia coli, purification of the recombinant coat protein and production of a polyclonal antiserum in rabbit. CP gene was found to be 780nt long, with a 256 deduced amino acid sequence encoding a predicted protein of 28 kDa. In filogenetic analysis, with RSPaV-SP and other variants of the virus, four groups were found and the sequence of RSPaV-SP showed the highest identity with the variant RSPaV-BS. The recombinant coat protein was purified by affinity chromatography and showed a molecular weight of 32kDa (4 kDa from a small vector sequence plus 28 kDa for the CP of RSPaV-SP). The antiserum proved specific for detection of the recombinant protein by Western Blot, and did not react with heterologous proteins starting at a dilution of 1:4000. At this dilution, the antiserum was effective in the virus detection of leaf extracts of infected plants and no reaction was observed with extracts from healthy grapevines. Considering that the virus is found at low concentrations in the plants during the seasons of the year, the results obtained so far were highly satisfactory for RSPaV detection. Serological methods have advantages over the biological indexing method, since they are cheaper and can be used in large-scale tests such as ELISA. Experiments using the ELISA technique were not successful. Purification of the native recombinant protein would be an alternative more efective to detect the virus using these technique.
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Ozimek, Paulina. "Pooling of rabbit antisera to reduce lot to lot variability of polyclonal antibodies." Thesis, KTH, Skolan för bioteknologi (BIO), 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-215021.
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Petit, Robert G. "The development of polyclonal antisera which inhibits purified Epstein-Barr virus (B95-8) DNA polymerase /." The Ohio State University, 1987. http://rave.ohiolink.edu/etdc/view?acc_num=osu148732966214544.
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Costa, Andréa Bernardes Vilhena. "Padronização da reação de Immuno-dot para detecção de Pet em sobrenadante de cultura de Escherichia coli enteroagregativa." Universidade de São Paulo, 2004. http://www.teses.usp.br/teses/disponiveis/9/9136/tde-31082009-163535/.
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Escherichia coli enteroagregativa (EAEC), destaca-se como um importante patógeno emergente causador de diarréia persistente em países em desenvolvimento e de diarréia aguda em países desenvolvidos. A grande heterogeneidade dos fatores de virulência caracteriza esta categoria, porém não foi estabelecido um marcador genético comum a todas as amostras de EAEC. O padrão de adesão agregativa (AA) em células HEp-2 e HeLa é a forma de caracterização e diagnóstico mais precisos desta categoria. Uma das toxinas envolvidas na patogênese é Plasmid-encoded toxin (Pet) pertencente à classe das proteínas autotransportadoras com características de uma serino protease denominada SPATEs. Iniciou-se este estudo com a determinação do padrão de adesão de 164 amostras EAEC, previamente caracterizadas como sonda pCVD432 ou onda AA positiva. Assim, 141 (86%) amostras, que apresentaram padrão de adesão agregativo, foram caracterizadas como EAEC. Face aos resultados obtidos, confirmou-se a baixa especificidade da sonda AA. A pesquisa do gene pet, por meio de ensaio de PCR, resultou na positividade de 12 (8,5%) amostras. Prosseguiu-se esse estudo com a padronização da reação de immuno-dot. Utilizando-se 300 µL do sobrenadante bacteriano, soro policlonal anti-Pet e o conjugado nas diluições 1/50 e 1/2.500, respectivamente, resultados bastante reprodutíveis foram obtidos. O método foi mais sensível que a detecção do gene por PCR. Por esse ensaio, detectou-se a toxina Pet em 16 (11,3%) das 141 amostras EAEC. Nenhuma das amostras controle negativo foi reconhecida pelo soro anti-Pet, assim como as amostras de E. coli produtoras das mais diversas toxinas. Apesar da baixa prevalência de amostras de EAEC produtoras da toxina Pet, neste estudo padronizou-se um método rápido, sensível, específico e de baixo custo para pesquisa desta toxina mostrando o potencial diagnóstico deste ensaio para uso em inquéritos epidemiológicos, o que poderá permitir determinar o papel da Pet no desenvolvimento de diarréia aquosa.<br>Enteroaggregative Escherichia coli (EAggEC) is an emerging diarrheal pathogen, whose pathogenesis is thought to comprise colonization of the intestinal mucosa with the release of secretogenic toxins. One of the toxin involved is the plasmid-encoded toxin (Pet), which is secreted by the autotransporter mechanism and belongs to a growing class of Enterobacteriaceae autotransporter proteins. Since the characteristic aggregative adherence pattern of EAggEC is associated with the presence of a large plasmid called pCVD432, DNA probes and PCR primers derived from this plasmid have been recommended as a screening method for EAggEC in the clinicai laboratory. In this study 164 E. coli isolates positive for the pCVD432 probe were tested for adherence to HEp-2 cells in which 141 isolates showed aggregative pattern, 12 isolates from them amplify a 1037-bp DNA fragment corresponding to pet gene by PCR. Using this samples we standardized an immuno-dot assay for EAggEC detection through Pet toxin as target antigen. 300 µl of bacterial supernatant were applied in a PVDF membrane, and using a rabbit polyclonal sera anti-Pet the expression of the toxin by immuno-dot was in the same isolates in which the gene was detected. Besides no negative controls reacted with Pet antisera, in which we included 40 isolates with no virulence markers for diarrheagenic E. coli and E. coli expressing toxins other than Pet. This method proves to be rapid, sensitive, specific and low cost, demonstrating this potential as diagnosis for Pet expression and its association with diarrhea.
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Hsu, Chia-wei, and 徐嘉偉. "Production and characterization of polyclonal antiserum against DC2 protein." Thesis, 2005. http://ndltd.ncl.edu.tw/handle/52383805355677344000.
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碩士<br>國立中山大學<br>生物科學系研究所<br>93<br>The hsDC2, an unknown gene, was located on chromosome 4q25. The genetic protein product contained 149 amino acids with the molecular weight of 16.8 kDa.
By bioinformatics, three predicted non-membrane portion of hsDC2 gene were designed to immune rabbit. The hsDC2 a1~31and hsDC2 a54~82 gene were inserted in pGEX6p-1 that expressed as fusion protein with GST. The other, hsDC2 a140~149, was synthesized with additional cycteine for conjugation with mcKLH at N-terminal. After four times subcutaneous injection, the antiserum was blooded from rabbits. The antibody was purified from the antiserum by protein A beads and confirmed with antigen by western blotting. The identified antibody was used to examine the expression of hsDC2 protein during U937 differentiation induced by RA and PMA.
Book chapters on the topic "Polyclonal antiserum AS"
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Bhat, Alangar Ishwara, and Govind Pratap Rao. "Production of Polyclonal Antiserum." In Springer Protocols Handbooks. Springer US, 2020. http://dx.doi.org/10.1007/978-1-0716-0334-5_28.
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Pihl, Tina H., Kristin E. Illigen, and Gunnar Houen. "Polyclonal Peptide Antisera." In Methods in Molecular Biology. Springer New York, 2015. http://dx.doi.org/10.1007/978-1-4939-2999-3_11.
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Bailey, Graham S. "Raising of Polyclonal Antisera." In Springer Protocols Handbooks. Humana Press, 1996. http://dx.doi.org/10.1007/978-1-60327-259-9_120.
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Green, Jonathan A., and Margaret M. Manson. "Production of Polyclonal Antisera." In Immunochemical Protocols. Humana Press, 1998. http://dx.doi.org/10.1007/978-1-59259-257-9_1.
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Weston-Kirkegaard, S., J. M. Souhrada, and H. F. Polesky. "Allotyping By Immunoblot Using Polyclonal Antisera." In Advances in Forensic Haemogenetics. Springer Berlin Heidelberg, 1990. http://dx.doi.org/10.1007/978-3-642-75496-8_59.
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Baker, Susan C., Amornrat Kanjanahaluethai, Nathan M. Sherer, David D. Axtell, and Jennifer J. Schiller. "Exploiting DNA Immunization to Generate Polyclonal Antisera to Coronavirus Replicase Proteins." In Advances in Experimental Medicine and Biology. Springer US, 2001. http://dx.doi.org/10.1007/978-1-4615-1325-4_44.
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Prager, E. M. "Polyclonal antisera elicited by lysozymes: Insights into antigenic structure and evolution." In Experientia Supplementum. Birkhäuser Basel, 1996. http://dx.doi.org/10.1007/978-3-0348-9225-4_14.
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Richter, J. "Polyclonal reference antisera may be useful for the differentiation of potyvirus species." In Potyvirus Taxonomy. Springer Vienna, 1992. http://dx.doi.org/10.1007/978-3-7091-6920-9_7.
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Tuberty, Shea R., Sergio F. Nates, and Charles L. McKenney. "Polyclonal Antisera Against Estuarine Crustacean Vitellins: A Molecular Approach to Reproductive Endocrinology and Toxicology." In Modern Approaches to the Study of Crustacea. Springer US, 2002. http://dx.doi.org/10.1007/978-1-4615-0761-1_5.
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Reis, V. M., M. Schloter, F. B. Reis, et al. "Characterization of Different Polyclonal Antisera to QuantifyHerbaspirillum spp. in Elephant Grass (Pennisetum purpureum Schun.)." In Biological Nitrogen Fixation for the 21st Century. Springer Netherlands, 1998. http://dx.doi.org/10.1007/978-94-011-5159-7_264.
Full textConference papers on the topic "Polyclonal antiserum AS"
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Dahlbäck, B., and A. LundWall. "ISOLATION AND CHARACTERIZATION OF cDNA CLONES FOR HUMAN FACTOR V." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643886.
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Coagulation factor V is a single chain, 330 kDa glycoprotein functioning as a cofactor to factor Xa in the activation of prothrombin. Thrombin cleaves factor V into four major fragments, out of which the N-terminal (105kDa) and the C-terminal (71-74kDa) fragments together constitute the active factor V species. To isolate cDNA clones a λ-gt 11 liver library was screened with a polyclonal, monospecific antiserum against human factor V. Four positive clones (two "weak", Aland A2 and two "strong", A3 and A4) were identified and isolated. Al(0.7kb), A2 (1.25kb) and A4 (0.85kb) reacted strongly with an antiserum against the 105 kDa, N-terminal fragment (heavy chain of factor Va), whereas A3 (1.25kb) gave the best signal with an antiserum against the 71-74 kDa, C-terminal fragment (light chain of factor Va). A1 hybridized with A2 and A4, whereas A2 only hybridized with Al. A3, which did not hybridize to any of the other clones, was used to rescreen the library and 9 positive clones (Bl-9) were isolated. B9 (3kb) coded for the entire C-terminal factor V fragment and the 3' noncoding sequence. B8 (1.8kb) partially overlapped B9 but extented the 5' sequence with 0.8kb. In a third screening round Al was used in combination with B8 and a 1.1 kb clone (CIO) was identified which hybridized to both. C10 did not hybridize with A2. The following overlapping cDNA clones can be orderedfrom the 5´end: A2-A1-C10-B8-B9 and together they cover 6 kb of coding sequence
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Bini, A., R. Mesa-Tejada, J. Fenoglio, B. Kudryk, and K. L. Kaplan. "SPECIFIC DETECTION OF FIBRIN IN HUMAN TISSUES BY A NEW IMMUNOHISTOCHEMICAL TECHNIQUE." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643316.
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Human biopsy (30), surgical (50) and autopsy (14) specimens of different embryonic origin (skin, blood vessel, kidney, lymph nodes, prostate, lung, liver, and intestine) were stained by the avidin-biotin complex immunoperoxidase technique (ABC-IP) with monoclonal antibodies (MAbs). MAb T2G1 (recognizes 315-42 and detects fibrin II in tissues), MAb I8C6 (recognizes BS1-42 and indicates fibrinogen and fibrin I), MAb GC4 (specific for fragments D and D-D), and a polyclonal antiserum for fibrinogen. The method can be applied to frozen or Boilin’s fixed paraffin embedded tissues with good preservation of morphology and high sensitivity at the light microscopy level. The results were compared with conventional histochemical stains currently used in surgical pathology to demonstrate fibrin deposits in tissues. These stains are based on the acidophilic properties of fibrin (Fraser-Lendrum for “more recent” and Mallory’s PTAH for “older” fibrin). ABC-IP staining with MAb T2G1 clearly detected fibrin in areas where Lendrum and PTAH failed to reveal fibrin deposits, e.g., in the intercellular and pericellular matrix, as well as in areas where staining occurred with conventional techniques, indicating greater sensitivity of the ABC-IP method. Fibrin was specifically detected in strands or clumps in some areas of inflammation and granulation tissue and seemed to be associated with platelets and macrophages. Moreover, ABC-IP with MAb I8C6 and MAb GC4 permits the distinction between fibrinogen or fibrin I, and D and D dimer which are poorly reactive with the Lendrum and PTAH methods. The polyclonal antiserum for fibrinogen showed reactivity with all the material stained with the MAbs and with some additional areas due to the epitopes of fibrinogen and fibrin not detected by the monoclonals. The ABC-IP technique with MAbs allows specific demonstration of fibrin deposits in tissues. Moreover, these results indicate that this method facilitates the correlation of the morphologic appearance of fibrinogen) -related deposits in tissues with their molecular form and will be useful to elucidate the role of fibrin in diseases such as atherosclerosis, kidney disease, and tumors.
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Van Nostrand, W. E., and P. P. Cunningham. "PROTEASE NEXIN II IN HUMAN PLATELETS." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643490.
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Normal human fibroblasts secrete a protein into the culture medium named protease nexin II (PN II), which forms sodium do-decyl sulfate (SDS)-stable complexes with epidermal growth factor binding protein (EGF BP). These complexes then bind back to the same cells and are rapidly internalized and degraded. We recently purified PN II to apparent homogeneity and showed that it is a single chain polypeptide with an estimated molecular weight of 106 KDa. Other interesting properties of this protein include its ability to bind heparin and its stability to treatment with SDS or pH 1.5. In addition to EGF BP, PN II will also complex the gamma subunit of 7S nerve growth factor (NGF-gamma) and trypsin. Here we show that human platelets contain a protein which possesses the same properties as does PN II from cultured human fibroblasts. This platelet PN II (PL-PN II) is also a 106 KDa single chain polypeptide which can complex EGF BP, NGF-gamma and trypsin. PL-PN II also possesses the unusual stability to treatment with SDS or pH 1.5. In addition, both PN II and PL-PN II exhibit the same affinity for binding to heparin-Sepharose. Furthermore, rabbit polyclonal antiserum raised against PN II recognized PL-PN II on Western blot analysis demonstrating that both proteins are immunologically related. Treatment of fresh unactivated platelets with epinephrine or thrombin resulted in a release of PL-PN II into the supernatant suggesting that PL-PN II is stored in the platelet alpha granules.
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Mannhalter, C., and E. Deutsch. "IMMUN0BL0TTING STUDIES OF THE INTERACTION OF PLASMA-FACT0R XI WITH KAOLIN." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644806.
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An immunoblotting technique with a commercially available, polyclonal Factor XI antibody was developed for Factor XI in plasma samples.Aliquots containing equivalents of 6 ul plasma were treated with appropriate amounts of SDS, electrophoresed on 7.5 % SDS-PAGE and immunoblotted onto nitrocellulose membranes. The blots were quenched with low fat milk and incubated with 2 % Factor Xl-antiserum, which was preadsorbed with Factor XI deficient plasma. The bound Factor XI antibody was identified by incubation with 125I-labelled Factor XI followed by autoradiography. Using this method, the interaction of Factor XI with kaolin was studied in plasma. Normal plasma was incubated with kaolin for various time periods. Aliquots were removed, centrifuged and the plasma supernatant and the kaolin pellets were examined after treatment with SDS on 7.5 % SDS-PAGE followed by immunoblotting.Factor XI was adsorbed onto kaolin and, after five minutes no Factor XI antigen was present in the plasma supernatant.However, after twenty minutes a band corresponding to Factor XI became again visible in the plasma, indicating a release of Factor XI from the kaolin surface. The electrophoretic results obtained with the SDS-eluates of the kaolin pellets confirm these observations. After thirty seconds a significant amount of Factor XI was present on the kaolin, and it exhibited the same electrophoretic mobility as Factor XI in the starting material. After ten minutes incubation, the protein concentration adsorbed onto the kaolin had increased. However, after twenty minutes clearly less Factor XI was present on the kaolin than after ten minutes.Thus, our results indicate that Factor XI is quickly adsorbed from plasma onto kaolin. However, Factor XI or XIa do not remain surface-bound but are released into solution.
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El-Badry, Nader, Safwat El-Haddad, and Ahmed Hussien. "Production and Evaluation of Polyclonal Antisera for Detection of Ralstonia solanacearum." In Proceedings of the II International Conference on Environmental, Industrial and Applied Microbiology (BioMicroWorld2007). WORLD SCIENTIFIC, 2009. http://dx.doi.org/10.1142/9789812837554_0018.
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Niewiarowski, S., A. Eckardt, H. Lukasiewicz, K. Norton, Tur-Fu Huanq, and E. Komecki. "PRODUCTS OF LIMITED PROTEOLYSIS OF GLYCOPROTEIN Ilia ON TEE PLATELET MEMBRANES." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643530.
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Previous investigations from our laboratories have demonstrated the limited proteolysis by chymotrypsin of GPIIIa and the formation of a major component migrating on SDS polyacrylamide gel electrophoresis with an apparent molecular weight of 60 kDa in a nonreduced system and 66 kDa in a reduced system. The formation of this component occurred in parallel with the express ion of fibrinogen receptors on the platelet surface. We raised in rabbits a monospecific polyclonal antibody against highly purified 60 kDa ccmponment. Using available rabbit polyclonal antisera (anti-GPIIIa and anti-66 kDa) and murine monoclonal anti GPIIIa antisera (AP3 and SSA6) a caiplete imnunological crossreactivity between GPIIIa and 60 kDa was established. The limit of detection by immunoblotting assay was 20 ng of GPIIIa or 60 kDa in Triton X100 extract of platelets. Extracts of intact platelets, of AEP- or thranbin-stEmulated platelets separated on SDS PAGE showed a strong band corresponding to GPIIIa, a light band migrating with an apparent molecular weight of 120 kDa and no detectable 60 kDa ccnponent. Incubation of platelets with chymotrypsin, pancreatic elastase or human granulocyte elastase resulted in the appearance of 60 kDa and in a progressive increase of 120 kDa component detectable by all polyclonal and monoclonal antisera. In contrast to 120 kDa, reduced GPIIIa (108 kDa) was not recognized by AP3 and SSA6. The 120 kDa did not dissociate in the presence of 6M urea and 5 mM EDTA. The component migrating with an apparent molecular weight of 120 kDa also appeared during prolonged storage of purified 60 kDa ccnponent. The partial purification of 60 kDa and 120 kDa components together with GPIIIa was accomplished by Con A sepharose chromatography. In conclusion 60 kDa and 120 kDa platelet membrane components represent products of proteolysis of GPIIIa.
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Rickles, F. R., W. W. Hancock, K. Kobzik, N. Hogg, and C. O’Hara. "THE DISTRIBUTION OF CROSS-LINKED FIBRIN IN HUMAN LUNG CARCINOMA PARALLELS THAT OF ACTIVATED HOST MONONUCLEAR LEUKOCYTES: IMMUNO-HISTOLOGIC STUDIES WITH MONOCLONAL ANTIBODIES." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643669.
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Previous immunohistologic studies of human lung carcinoma, using polyclonal antibodies to antigens shared between fibrinogen (FGN) and fibrin (FB), showed that FGN/FB were associated withtumor cells. These findings were interpreted as evidence of the presence of tumor-associated procoagulant activity(PCA). Wecompared the distribution of coagulation-associated proteins in 16 casesof human carcinoma of the lungofvarying histologic types, using polyclonal antibodies to FGN/FB and monoclonal antibodies (mAb) to cross-linked fibrin (XL-FB;UC-45), fibronectin (FN;PHM13), factor VIII (vWF:Ag)and a tissue factor-related antigen(TF:RAg;A1 -3)- Host mononuclear leukocytes were identifiedusing various mAb toT cells and macrophages, and studied for their expression of receptorsfor interleukin-2 (IL-2R). Positive resultsare summarizedIn addition, studies of the mononuclearcells adjacent to tumors in 12/12 casesshowed the presence of tumor-associated macrophages, 10/11 showed T cells,mainly T8+, and A/5 showed corresponding expression of IL-2R, suggesting cell activation.The use of highly specific mAb showed that XL-FB is actually more selectively distributed than is found using polyclonal antisera to FGN/FB, and indeed XL-FB was largely confined to those areas adjacent to tumors which are rich in mononuclearcells. These findings suggest that fibrin deposits in human carcinomasof the lung may be due todevelopment of PCA by activated host mononuclear cells, rather than tumor cells.This lack of XL-FB on tumor cellsinspite of A1- 3 binding suggests that TF:RAg may not be available on the tumor cell surface for the activation of clotting. Further studies are neededto define the functional capacity of PCA molecules on tumor cells and tumor-associated mononuclear cells in situ.
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Colman, R. W., A. Gewirtz, D. L. Wang, et al. "BIOSYNTHESIS AND EXPRESSION OF FACTOR V IN MAGAKARYOCYTES." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1642955.
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Coagulation factor V (FV), is a single chain, multifunctional glycoprotein of Mr 350,000 which interacts with a variety of hemostatic proteins such as factor Xa, prothrombin, thrombin and protein C, on the surface of platelets and vascular endothelial cells. FV serves as both a cofactor and substrate in the generation of thrombin and plays a critical regulatory role in both physiologic hemostasis and pathologic thrombosis. The biosynthesis of FV and its subsequent expression are therefore expected to be precisely controlled and may differ in the three sites of synthesis - hepatocytes, endothelial cells, and megakaryocytes (MK). We have previously demonstrated that each guinea pig MK contains 500 times as much FV as in a platelet, as quantified by a competitive enzyme-linked-immunosorbent assay and expresses FV by cytoimmunofluorescence. De novo biosynthesis was demonstrated by incorporation of S-methionine into FV purified on a immunoaffinity column. The purified MK protein exhibited both FV coagulant activity and antigenicity. However, MK FV was more slowly activated by thrombin, more stable in the absence of Ca and exhibited a slightly higher M of 380,000 compared to plasma FV. Similar studies have documented biosynthesis in human MK. In addition, all morphologically recognizable MK enriched by elutriation from human bone marrow contained FV as documented by both monospecific polyclonal and monoclonal antibodies (MAb) to FV. All these cells bound FV since a murine MAb reacting with the light chain of FV (B38) labeled all cells. In contrast, 68% of cells synthesized FV since B10, a MAb to the activation peptide recognizing FV but not FVa, labeled this fraction. To determine whether immature nonnorphologically recognizable MK expressed FV, we identified these cells with an antiserum to human platelet glycoproteins and then probed them with B38. Seventy percent (70%) of such small cells expressed FV. In contrast, no small cells in MK colonies cloned in FV deficient medium expressed FV while only 40% of such colonies contained cells which expressed FV.To further probe the regulation of FV in MK we attempted to correlate the synthesis of FV as probed by MAb B10 with geometric mean cell diameter, stage and ploidy. No significant correlation of FV with any of these indicators of MK maturation. In contrast, preliminary studies suggest that low doses of tetradecanoyl phorbol acetate augment both the number of MK containing FV and the level of FV expressed by individual cells. Thus, FV synthesis may be regulated independent of size, stage, or ploidy and protein kinase C may play a role.To further define the molecular nature of FV in MK we found that purified FV was converted from a monomer to high Mr multimers by an enzyme derived from MK. These multimers resulting from covalent crosslinking since they were stable to SDS, 100° C and reducing agents. The responsible enzyme appeared to be MK FXIIIa since it required C, was inhibited by agents which react with the active site thiol group and was blocked by pseudoamine donor substrates such as putrescine. In addition, FXIIIa was directly demonstrated in guinea pig MK by a specific activity stain. Other investigators have established that FV became irreversibly associated with platelet cytoskeletons after exposure to thrombin. tested whether FXIIIa might mediate this association by performing ligand blotting of platelet membrane proteins using 125I-FV(FV*). Only actin of all the membrane proteins was detected by radioautography. The binding of FV* to the cytoskeleton was dependent in the presence of Ca and FXIIIa. In purified systems crosslinked complexes containing FV* or radiolabeled actin were detected in separate experiments. In whole platelets, the formation of the heteropolymer, after thrombin stimulation, was inhibited by antibodies to FXIII a chain, FV activation peptide (B10) or actin. Endogenous platelet FV was also dependent on FXIII for incorporation into the platelet cytoskeleton after thrombin stimulation. When thrombin-treated FV was crosslinked to actin only the activation peptide (150 kDa) was crosslinked. The light chain or heavy chain of FVa were not involved. Thus FXIIIa play an important role in the binding of FV in platelets to the cytoskeleton during activation and secretion.Further studies of FV in megakaryocytes are necessary to define the regulation of biosynthesis and the control of expression which dictate its critical role in hemostasis and thrombosis.
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Uzan, G., A. Lajmanovich, M. H. Prandini, Ph Frachet, A. Duperray, and G. Marguerie. "MOLECULAR CLONING OF PLATELET GPIIb FROM HEL CELLS AND HUMAN MEGAKARYOCYTES." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643960.
Full textAbstract:
Platelet GP IIb-IIIa is an heterodimer which functions as a receptor for fibrinogen, fibronectin and Von Willebrand factor and is implicated in platelet adhesive reactions. To study the structure function relationship of this glycoprotein, a recombinant DNA approach was initiated. cDNA expression libraries were constructed in » gtll vector, from erythro-leukemia cells (HEL) and megakaryocytes mRNA. The human megakaryocytes were isolated from patients with chronic myeloid leukemia. The HEL library was initially screened with polyclonal antibodies anti GPIIb IIIa. One clone, λIIbI, containing a 1.65 kbp insert reacted with a panel of different polyclonal antibodies anti GPIIb IIIa and a monoclonal antibody anti GPIIb. To further characterize this clone the synthesis of the fusion protein was induced by IPTG. The bacterial protein was then blotted onto nitro cellulose and incubated with antisera anti GPIIb-IIIa. Antibodies that specifically bound with the fusion protein were eluted and tested on platelet membrane extracts. The selected antibodies produced a positive signal at the GPIIb position similar to the signal produced by the monoclonal antibody anti GPIIb on the same membrane extract. Finally on western blotting, a protein of Mr= 170kD reacted with the monoclonal antibody anti GPIIb. λIIbI insert was used to screen the megakaryocyte library and 3 clones, λIIb2,λIIb3 and λIIb4 were isolated. The size of HEL cells and megakaryocytes GPIIb mRNA was estimated by northern blotting. Only one species of 3.9 kb was identified in both cells. The four different clones accounted for 50% of the coding sequence of this mRNA.Sequencing of these cDNAs indicated that the plasmatic domain of GPIIb contains a cystein rich region. The sequence of these clones will allow the study of the adhesines genetic diversity in different cellular systems.
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Fulcher, C. A., R. A. Houghten, S. de Graaf Mahoney, J. R. Roberts, and T. S. Zimmerman. "SYNTHETIC PEPTIDE PROBES OF FACTOR VIII IMMUNOLOGY AND FUNCTION." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644768.
Full textAbstract:
In order to develop specific immunologic reagents for mapping functionally important sites on FVIII, we have prepared rabbit polyclonal antibodies against synthetic peptides of FVIII derived from regions along the entire FVIII amino acid sequence. To date, a total of 70 peptides have been synthesized and characterized by amino acid and HPLC analysis. The peptides were coupled to keyhole limpet hemocyanin with glutaraldehyde as a linkage reagent and used to immunize rabbits. Antisera were tested by ELISA assay on polystyrene microtiter plates coated with either the peptide immunogen, or purified FVIII. The antisera were also tested for their ability to inhibit FVIII clotting activity and to react with separated FVIII polypeptides on immunoblots.Of the 70 peptides, all reacted with the peptide immunogen, 45 reacted with purified FVIII and 33 reacted with FVIII on immunoblots. Because we had obtained evidence that cleavage of the amino terminal region of the 80 kDa polypeptide may play a role in FVIII activation by thrombin, a series of partially overlapping peptides, 15 residues in length, were synthesized in this area. After affinity purifying these antibodies on columns of FVIII immobilized on agarose, adjusting the antibodies to equal antigen binding titers by dot immunoblotting and testing for inhibition of FVIII activity, only one antibody could strongly inhibit FVIII clotting activity. This inhibition could be blocked by the peptide itself at nanomolar concentrations and no significant inhibition could be shown by antibodies to partially overlapping peptides individually, or in combination. These data suggest that a site important to FVIII function can be localized to a 15 amino acid residue region of the 80 kDa polypeptide of FVIII. In addition, a second inhibitoryantibody was identified which was produced against a peptide in the carboxy terminal region of the 54 kDa thrombin fragment of FVIII and this area is currently being studied in a similar manner. In addition, two monoclonal anti-FVIII synthetic peptide antibodies have been produced which react with purified FVIII on immunoblots. One of these antibodies also functions as an immunoadsorbent when linked to agarose and FVII can be purified in this manner, using the synthetic peptide as eluant. It is evident that antibodies to synthetic peptides of FVIII can be useful probes of FVIII structure, function and interactions as well as being of use in FVIII purification.