Enzyme Linked Immunosorbent Assay

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Development of an enzyme linked immunosorbent assay for detection of cyathane diterpenoids

Development of an enzyme linked immunosorbent assay for detection of cyathane diterpenoids

Results: An indirect competitive enzyme linked immunosorbent assay has been developed for detection of cyathane type diterpenoids. Rabbit polyclonal antibodies were raised against a mixture of striatal A and B conjugated to bovine serum albumin. The conditions for direct attachment of the hapten striatal B to a solid phase by passive adsorption were optimized. The cross reactivities of the striatals A, C and D, of the striatins A and B, and of the erinacines C and P to striatal B were determined. The validation study showed that the ELISA was precise and sensitive. The average IC 50
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Entwicklung und Anwendung eines Enzyme-Linked-Immunosorbent-Assay (ELISA) für die serologische Erfassung der Campylobacter-Siuation in Schweinebeständen mittels Blutserum- und Fleischsaftproben

Entwicklung und Anwendung eines Enzyme-Linked-Immunosorbent-Assay (ELISA) für die serologische Erfassung der Campylobacter-Siuation in Schweinebeständen mittels Blutserum- und Fleischsaftproben

Tabelle 89 Darstellung der OD-Werte von 1:30 und 1:100 verdünnten Feldfleischsaftproben (FS 24) auf einer mit C.c./C.jej.-Mischantigen (Mix) beschichteten Platte sowie der Ergebnis[r]

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Clinical validation of a novel enzyme-linked immunosorbent spot assay-based in vitro diagnostic assay to monitor cytomegalovirus-specific cell-mediated immunity in kidney transplant recipients: a multicenter, longitudinal, prospective, observational study

Clinical validation of a novel enzyme-linked immunosorbent spot assay-based in vitro diagnostic assay to monitor cytomegalovirus-specific cell-mediated immunity in kidney transplant recipients: a multicenter, longitudinal, prospective, observational study

monitor the response of patients to immunosuppression. On the other hand, IFN-c ELISpot test results were increased in most patients following CMV reactivation, as expected from the reactivation of CMV-specific memory T cells. Increased pp65-specific CMI after but not at visits affected by CMV events, as opposed to IE-1-specific CMI, might reflect the kinetics of CMV reactivation, IE-1 being an immediate-early gene and acknowledged marker of early CMV reactiva- tion as opposed to the late antigen pp65 [10,58,59]. It might additionally reflect the dynamics of IE-1- and pp65-specific CD8 + and CD4 + T-cell responses during CMV infection and reactivation [6–8,10]. That most patients were responders in the ELISpot assay correlated nicely with the absence of clinical outcome in this cohort, further suggesting that the majority of patients still presented a protective immunity against CMV. Likewise, this finding indicates that the IFN-c ELISpot assay might identify nonresponders as patients poten- tially over-immunosuppressed. Conversely, ELISpot test results were increased prior to graft rejection. As 13 of 15 (87%) of graft rejection episodes were attributed to or suspicious for TCMR, the ELISpot assay might be able to monitor strong cellular immunity in potentially under-immunosuppressed patients. The absence of stan- dardized immunosuppressive therapy in this study did not allow to directly address whether immunosuppres- sant doses correlated to the occurrence of infections and graft rejections. Finally, changes in CMV-CMI in relation to occurrence of opportunistic infections were similar to those detected in association with CMV- related complications, indicating that IFN-c ELISpot results, notably in response to pp65, might not only reflect CMV-CMI but more globally the patient’s
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Clinical validation of a novel enzyme-linked immunosorbent spot assay-based in vitro diagnostic assay to monitor cytomegalovirus-specific cell-mediated immunity in kidney transplant recipients: a multicenter, longitudinal, prospective, observational study

Clinical validation of a novel enzyme-linked immunosorbent spot assay-based in vitro diagnostic assay to monitor cytomegalovirus-specific cell-mediated immunity in kidney transplant recipients: a multicenter, longitudinal, prospective, observational study

SFC was approximately 4 times higher at visits documented with CMV complications (44 vs. 12 SFC/ 200 000 cells; MWU P = 0.007; ANOVA on ranks P = 0.038), while median pp65-specific SFC levels were comparable at visits with and without CMV events (116 vs. 151 SFC/200 000 cells, respectively; MWU P = 0.497) (Fig. 3a). On the other hand, IE-1- and pp65-specific test results were increased in 11 of 14 (79%) and 12 of 14 (86%) of paired samples, respectively, following CMV complications, with median ratios after-to-before CMV complication of 1.8 (Wilcoxon signed-rank test, P = 0.009) and 2.4 (Wilcoxon signed-rank test, P = 0.007), for IE-1 and pp65, respectively (Fig. 3b). These observations suggest that the IFN-c ELISpot assay can monitor changes in CMV-CMI consecutive to manifestations of CMV com- plications, upon ex vivo stimulation of PBMC with IE-1 and pp65 antigens.
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Synthesis of Cross-Linked Polyamines 

Synthesis of Cross-Linked Polyamines 

In a n o th e r ap p ro ach to cross-linked polyam ines (Schem e 4), the prim ary am ino groups of the m odel com pound d ieth y len etriam in e (2) were p ro tected by the carbobenzoxy group using the N- (benzyloxycarbonyloxy) succinim ide re a g e n t (15). The cross-linking of the p ro te c te d 16 to give 20 was achieved w ith suberic acid (17) by an a d a p ­ tatio n of the eleg an t literatu re m eth o d [18] (Schem e 4) using phenyl fr/s-(2-thioxo-l,3-thiazoli- dine-3-yl)phosphine oxide (18) as the acyl tran sfer agent in the presence of the sterically h indered base N ,N -dim ethylcyclohexylam ine (19). The tran sfer agent 18 was p re p a re d from phenylphos- phonyl dichloride and 2 -m ercaptothiazoline in the
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Renal Aminoacylase, a Zinc Enzyme 

Renal Aminoacylase, a Zinc Enzyme 

In 1952 Greenstein and coworkers1 observed that the activity of renal aminoacylase is influenced by Co2+ and that the effects varied with the nature of the substrate: an activation as well as an in­ hibition was observed depending on the structure of the N-acylamino acids. These findings were con­ firmed in a later publication2 which revealed also that the enzyme is active in the absence of Co2+; no Co2+ could be detected in enzyme preparations which were isolated under Co2+ free conditions. The question wether aminoacylase is a real metallo enzyme or if the metal is only an effector which varies the activity of the metal free enzyme re­ mained obscure. Within the scope of our investiga­ tions on the catalytic mechanism and the physio­ logical function of aminoacylase3-6, we have also studied the effects of metal ions on the activity of the enzyme. In the present communication we report the results of these experiments, which have shown that aminoacylase is a zinc containing metallo enzyme.
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Neuartige Enzyme aus Basidiomyceten für technische Anwendungen

Neuartige Enzyme aus Basidiomyceten für technische Anwendungen

Auf der Suche nach neuen Enzymen für neue Aufgabenstellungen, sowie stabileren und effizienteren Enzymen für bestehende Aufgaben werden weiterhin verschiedenste Organismengruppen nach diesen durchgemustert und für die Biotechnologie erschlossen. Der Gruppe der Basidiomyceten (Ständerpilze), zu denen auch die in dieser Dissertation eingesetzten Pilze gehören, wurde lange Zeit nur am Rande Beachtung geschenkt. Zu Unrecht, denn die von ihnen ausgeschiedenen Enzyme weisen eine hohe Diversität an neuartigen enzymatischen Funktionalitäten auf. Ihre vergleichsweise hohe Stabilität gegenüber verschiedensten Umweltfaktoren, die unter anderem durch einen hohen Glycosylierungsgrad bedingt ist, macht sie für den großtechnischen Einsatz besonders interessant. Die Glycosylierung von Proteinen ist ein zwischen den einzelnen Eukaryotengruppen stark konservierter Prozess, der mehrere Funktionen erfüllt. Neben der Adressierung der Proteine für den intrazellulären Transportprozess dient er der korrekten Faltung im Endoplasmatischen Retikulum und der Vermeidung von Proteinaggregation. In Zusammenhang mit dieser Doktorarbeit ist besonders der Aspekt der mit der Glycosylierung verbundenen erhöhten Stabilität, sowie die mit ihr einhergehende Resistenz gegen einen einen Abbau durch Peptidase interessant. Biotechnologisch ist diese Modifizierung des Enzyms von hohem Interesse. Die Enzyme haben höhere Haltbarkeit, und vertragen größere Schwankungen bei pH-Wert, Temperatur, Druck und des Salzgehalts des Mediums. Auch Lösungsmittel können zum Teil in höheren Konzentrationen toleriert werden.
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Kopplung der Cyclooxygenase 2 an sekundäre Enzyme

Kopplung der Cyclooxygenase 2 an sekundäre Enzyme

Acetylsalicylsäure, bekannt als Aspirin, ist weltweit eines der am häufigsten eingenommenen Schmerzmittel und gehört zur Gruppe der nicht steroidalen Antirheumatika. Der Wirkmechanismus beruht auf der Hemmung zweier Enzyme, der Cyclooxygenasen 1 und 2 (Cox-1 und Cox-2), welche auch als Prostaglandin- Endoperoxid-H-Synthasen bezeichnet werden. Dadurch kommt es zu einer Hemmung der Bildung von Prostaglandinen, welche Schmerzmediatoren sind. Allerdings werden durch diese Hemmung in nicht unerheblichem Maße physiologische Wechselwirkungen gestört, so dass es zu Nebenwirkungen, wie zum Beispiel (z.B.) Magenschleimhautblutungen, kommen kann. In den letzten Jahren sind daher Medikamente entwickelt worden, die zwar die Schmerzen lindern, die bekannten Nebenwirkungen aber nicht mehr hervorrufen sollen (G IERSE et al. 1996). Das Resultat war eine Gruppe von Cox-2 selektiven
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Polysulfatierte Dendrone als Transportvehikel für Enzyme in der Tumortherapie

Polysulfatierte Dendrone als Transportvehikel für Enzyme in der Tumortherapie

Introduction: Conventional chemotherapeutic drugs are mostly associated with high systemic cytotoxicity because of their low selectivity. Selectivity can be increased by the use of targeted drug delivery systems which lead to lower side effects and better therapeutic outcomes. Dendritic polymers have shown to be promising targeted drug delivery systems (TDDS) in cancer. In this thesis, the MacroDel (Macromolecular Delivery), a synthetic polysulfated Dendron made of glycerol monomers, is evaluated regarding its ability to act as a TDDS for enzymes. Several enzymes have high cytotoxic potential. However, the inability to transport them to their cytoplasmatic target has been the major reason why enzymes have rarely been used in chemotherapies. By using the example of RNase, DNase and Asparaginase, this thesis analyses the potential of MacroDels as targeted drug delivery systems for enzymes in cancer treatment. Methods: The conjugation of the enzymes to the MacroDel was controlled through native gelelectrophoresis and the enzyme activity after conjugation through photometry. The MacroDel and the MacroDel-enzyme conjugates were conjugated to ICC-dyes. The in vitro uptake and distribution were then analysed on all cancer cell lines using flow cytometry and fluorescence microscopy. The in vivo analysis was performed on Nude-Fox1 nu mices transplanted with the human coloncarcinoma HT29 and a gastropancreatic tumor. Cytotoxicity of the MacroDel and its enzyme-conjugates were examined in vitro using MTT-assays. The uptake of the MacroDel and its conjugates were analysed by inhibiting OATP1B1 with Rifamycin.
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Biological Systems to Assay Herbicidal Bleaching 

Biological Systems to Assay Herbicidal Bleaching 

sensitivity during the greening process against the pyridazinone herbicide SAN 9789 at concentrations of about 10- 8 M (Fig. 1 A ). In this system linearity between reciprocal value of chlorophyll content and herbicide concentration is established and the Dixon plot [8 ] can be employed for cellular systems determ ining the I 50 as 8.8 x 10~9 M. The same fig­ ure can be arb itrarily estimated directly from the graph of Fig. 1 A. W ith concentrations of 5 x 10~8 M SAN 9789 and higher greening is inhibited com­ pletely. Due to the good linearity of the Dixon plot the assay using the greening process allows for the
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Linked Data Supported Information Retrieval

Linked Data Supported Information Retrieval

nence per dataset. A green (bold) label indicates the highest value and a red (italic) the lowest value in each category. Since not all en- tities can be linked with a type or affiliated with the ranking, the values for each partition do not necessarily sum up to 100%. For each dataset the percentage of entities per category is denoted, as e. g., of all the entities in the KORE50 dataset 47.1% are persons and 6.9% are places. As Steinmetz et al. [68] have demonstrated, there is a signif- icant number of untyped entities in the DBpedia Spotlight and the KORE50 datasets. Therefore, an extra row for unspecified entities has been added to the table. The first partition (row 1–4) can be consid- ered as an indicator of how specialized a dataset is. Thus, for the evaluation of an annotator with focus on persons, the KORE50 data- set with 45.1% of person annotations might be better suited than the IITB dataset with only 2.4% of person annotations. The second and third partition (PageRank and HITS) show the entities categorized ac- cording to their popularity. It can be observed that many datasets are slightly unbalanced towards popular entities. A well balanced data- set should exhibit a relation of 10%, 45%, 45% among the three subset categories.
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Is offshoring linked to offshoring potentials? Evidence from German linked-employer-employee data

Is offshoring linked to offshoring potentials? Evidence from German linked-employer-employee data

The effects are stronger in larger and exporting plants, but hold for alternative measures of offshoring potential like in Spitz-Oener (2006), Blinder (2009), or Becker et al. (2013). 24 Also, we show that a compound index capturing several dimensions of offshorability, as proposed by Brändle and Koch (2014), is preferable over the inclusion of the underlying job characteristics in such estimation. The contribution of the paper is that it combines rich information on offshoring potential of jobs from representative German task data to detailed information from German linked employer-employee data, a combination that has not been available so far. Also, this study constitutes the first attempt to analysing the link between offshoring and its potential at the micro-level, with the exception of Eppinger (2014). Most previous studies were concerned only with one of these phenomena and typically relied on industry-level information, such as shares of imported intermediates inputs from the WIOD.
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Molekulare Grundlagen und Anwendungsmöglichkeiten des Comet Assay in Pflanzen

Molekulare Grundlagen und Anwendungsmöglichkeiten des Comet Assay in Pflanzen

Bislang ist unklar, in welchem Umfang sogenannte „alkali-labile“ Stellen, z.B. abasische Stellen, die durch den Verlust von Basen, entweder durch spontanen Zerfall oder durch aktive Entfernung geschädigter Basen im Zuge der Basenexcisions-Reparatur entstehen, in den alkalischen Comet Assay-Varianten in DNA-Strangbrüche umgesetzt und detektiert werden können. Die betroffenen Zuckerreste gelten als anfällig für eine Hydrolyse der Phosphodiester-Bindung durch Alkali (pH > 12.8, Lindahl und Anderson, 1972). Durch eine zusätzliche Behandlung von Comet-Präparaten mit AP-spezifischen Endonukleasen konnte gezeigt werden, dass ein bedeutender Anteil an AP-Stellen im Comet Assay nicht detektiert wird (Fortini et al., 1996; Angelis et al., 1999).
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Interzelluläre Heterogenität strahleninduzierten DNS-Schadens im Comet-Assay

Interzelluläre Heterogenität strahleninduzierten DNS-Schadens im Comet-Assay

Zusammenfassung: Initialschadensheterogenität von Blutleukozyten im Comet- Assay ist zumindest zum großen Teil nicht Ergebnis interzellulär verschiedener Quantität des DNS-Schadens durch Differenzen der Antioxidantienkonzentration, sondern Produkt chromatinmodulierter quantitativer Schadensunterschiede bzw. der durch den DNS-Packungsgrad und vor allem den mittleren Schadensgrad beeinflussten Messbarkeit des Schadens mit dem Comet-Assay. Abschließend geklärt werden konnte nicht, ob Unterschiede des DNS-Packungsgrades bzw. der Chromatinstruktur zu einer veränderten Induktion von DNS-Schäden führten, oder ob diese Unterschiede eine veränderte Detektierbarkeit dieser Läsionen bedingen. Eine Trennung beider Effekte war experimentell nicht möglich.
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Entwicklung und Realisierung einer Strategie zur Syndikation von Linked Data

Entwicklung und Realisierung einer Strategie zur Syndikation von Linked Data

Nonnenstrasse 11d, D-04229 Leipzig, raphael.doehring@netresearch.de http://netresearch.de Abstract. Recently, the publishing of structured, semantic information as linked data has gained quite some momentum. For ordinary users on the internet, however, this information is not yet very visible and (re-) usable. With LESS we present an end-to-end approach for the syndi- cation and use of linked data based on the definition of templates for linked data resources and SPARQL query results. Such syndication tem- plates are edited, published and shared by using a collaborative Web platform. Templates for common types of entities can then be combined with specific, linked data resources or SPARQL query results and inte- grated into a wide range of applications, such as personal homepages, blogs/wikis, mobile widgets etc. In order to improve reliability and per- formance of linked data, LESS caches versions either for a certain time span or for the case of inaccessibility of the original source. LESS sup- ports the integration of information from various sources as well as any text-based output formats. This allows not only to generate HTML, but also diagrams, RSS feeds or even complete data mashups without any programming involved.
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Stabilization of Industry-Relevant Enzyme Formulations

Stabilization of Industry-Relevant Enzyme Formulations

This chapter deals with metal-organic frameworks (MOFs) as enzyme stabilizing agent in a liquid detergent. The general approach is displayed in figure 5-27 starting with co-precipitation for enzyme immobilization and encapsulation (enzyme ⊂ MOF). As described in chapter 2.7.1. co-precipitation is used to combine enzymes with MOFs due to the unsatisfied results obtained with presynthesized mesoporous silica nanoparticles. Enzyme and MOF precursors co-precipitate and the framework is built up around the enzyme in-situ (figure 5-27). After three washing cycles of the MOF-enzyme adduct, the MOF is characterized regarding crystallinity (PXRD) and crystal structure (TEM). Encapsulation efficiency is determined – if possible – using the BCA assay of the supernatant removed in the first washing cycle. It is assumed that enzyme molecules, which are not recovered in the supernatant are encapsulated in or immobilized onto the MOF. The enzyme activity of the redispersed washed MOF phase is measured to calculate the amount of active enzyme within the MOF. MOFs with promising results regarding encapsulation efficiency and enzyme activity are examined in storage tests in standard detergent formulations and in the case of lipase in aqueous solutions against protease degradation. Finally, lead candidates are investigated in washing tests whether the encapsulation or immobilization using MOFs has an influence on the enzymes´ performance towards standard stains on textiles (figure 5-27).
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Kinetische und strukturelle Untersuchung der Katalysemechanismen ausgewählter Kofaktor-abhängiger Enzyme - Implikationen für die Decarboxylierung von α-Ketosäuren durch Thiamindiphosphat-abhängige Enzyme

Kinetische und strukturelle Untersuchung der Katalysemechanismen ausgewählter Kofaktor-abhängiger Enzyme - Implikationen für die Decarboxylierung von α-Ketosäuren durch Thiamindiphosphat-abhängige Enzyme

Diese Beschleunigung stellt den ersten Mechanismus dar, nach dem in allen ThDP-abhängigen Enzymen eine Erhöhung der Reaktionsgeschwindigkeit im Vergleich zur Reaktion in Lösung gewähr[r]

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Untersuchungen zur Zytotoxizität mittels WST-I-Assay und zur Gentoxizität mittels Comet-Assay von Portlandkompositzementen mit unterschiedlichen Zusatzstoffen, sowie Klinker und Kalksteinmehl in humanen Lungenzellen

Untersuchungen zur Zytotoxizität mittels WST-I-Assay und zur Gentoxizität mittels Comet-Assay von Portlandkompositzementen mit unterschiedlichen Zusatzstoffen, sowie Klinker und Kalksteinmehl in humanen Lungenzellen

Neben den Mechanismen, die über Entzündungsreaktionen zur Tumorentstehung beitragen, werden auch Tumorursachen anderer Genese diskutiert. Es deutet sich an, dass bei Rauchern mit Quarzexposition ein erhöhtes Risiko für Lungenkrebs besteht. So lagen Überlegungen nahe, dass bestimmte Enzyme wie P450 1A1 (= CYP1A1), die in die Kanzerogenese des Rauchens eingebunden sind, auch von Quarz beeinflusst werden können. CYP1A1 spielt eine große Rolle bei der Umwandlung von polycyclischen Aromaten, wie z.B. Benzo(a)pyren aus Zigarettenrauch in krebsinduzierende Epoxide. Die Hypothese konnte unterstützt werden, indem man in vitro in Rattenlungenzellen nach Quarzexposition eine dosis-abhängige Induktion von CYP1A1-mRNA und in vivo eine Erhöhung der Expression des Aryl-Hydrokarbon- Rezeptors beobachtet hat und zum Entschluss kam, dass die Effekte von Quarz als kokarzinogen gesehen werden können.(Becker et al., 2006)
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Assay-Entwicklung und Inhibitorsynthese für die Trans-sialidase aus Trypanosoma cruzi

Assay-Entwicklung und Inhibitorsynthese für die Trans-sialidase aus Trypanosoma cruzi

In this thesis new C-glycosidic derivatives of sialic acid were synthesized as potential inhibitors or modulators of enzyme activity, based on the known crystal structure of TcTS in complex with sialyl lactose. The main goal was to address the acceptor binding site, composed of two aromatic amino acid residues. This site is responsible for the trans- sialylation reaction in preference to simple hydrolysis of sialosides. Binding to the acceptor binding site was accomplished by including aromatic residues in the newly synthesized ligands. These residues should mimic the hydrophobic interaction between galactosides and the aromatic amino acid side chains by enabling aromatic π-stacking. The synthetic method of choice was primarily the ruthenium catalyzed cross metathesis of allyl C-glycosides with derivatives of styrene and sugar olefins.
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Potenziale von Linked Open Data in Öffentlichen Bibliotheken

Potenziale von Linked Open Data in Öffentlichen Bibliotheken

Let me first bring you up to date on how far we have go6en so far. The library currently runs a project which will last un7l ca. 2020, when the new main public library in Oslo is due to open (the opening has been postponed several 7mes). This project is responsible for developing and acquiring so;ware applica7ons for future library services. Currently the project is working on a new library system, which is based on the open source system Koha, but that has separate extensions for cataloguing and end-user services such as search, to use RDF linked data as a basis. The cataloguing module and the new search service are being developed right now, so the library hasn’t actually started using this technology in everyday work yet. I am therefore unable to answer your ques7ons on the outcome, 7me spent on cataloguing etc.
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