TY - JOUR A1 - Schenk, Jörg A. A1 - Fettke, Jörg A1 - Lenz, Christine A1 - Albers, Katharina A1 - Mallwitz, Frank A1 - Gajovic-Eichelmann, Nenad A1 - Ehrentreich-Förster, Eva A1 - Kusch, Emely A1 - Sellrie, Frank T1 - Secretory leukocyte protease inhibitor (SLPI) might contaminate murine monoclonal antibodies after purification on protein G JF - Journal of biotechnology N2 - The large scale production of a monoclonal anti-progesterone antibody in serum free medium followed by affinity chromatography on protein G lead to a contamination of the antibody sample with a protein of about 14 kDa. This protein was identified by mass spectrometry as secretory leukocyte protease inhibitor (SLPI). This SLPI contamination lead to a failure of the fiber-optic based competitive fluorescence assay to detect progesterone in milk. Purification of the monoclonal antibody using protein A columns circumvented this problem. KW - Hybridoma KW - SLPI KW - Protein G KW - Progesterone KW - Serum-free Y1 - 2012 U6 - https://doi.org/10.1016/j.jbiotec.2011.12.025 SN - 0168-1656 VL - 158 IS - 1-2 SP - 34 EP - 35 PB - Elsevier CY - Amsterdam ER - TY - THES A1 - Sellrie, Frank T1 - Immuntechnologische Verfahren zum Aufbau homogener Immunoassays sowie zur Selektion Antikörper produzierender Zellen T1 - Immunotechnological procedures for the development of homogeneous immunoassays and the selection of antibody producing cells N2 - Homogene Immunoassays sind immunologische Testverfahren, bei deren Durchführung vollständig auf Separations- und Waschschritte verzichtet werden kann. Der Substrate Channeling Immunoassay beruht auf der Weitergabe eines Substrates in einem immunologischen Komplex aus zwei Enzymen. Das Produkt des ersten Enzyms dient dem zweiten Enzym als Substrat zur Generierung eines photometrisch nachweisbaren Produktes. Voraussetzung für diese Weitergabe ist die enge räumliche Nähe beider Enzyme. Diese Nähe wird durch eine Bindung zwischen Analyt und anti-Analyt Antikörper vermittelt. Ein solcher Substrate Channeling Immunoassay wurde unter Verwendung der Enzyme Glucoseoxidase und Peroxidase aufgebaut. Das so etablierte System war funktionstüchtig, jedoch blieb seine Sensitivität hinter der normaler, heterogener Immunoassays zurück. Die Grundlage eines Fluorescence Quenching Immunoassays ist der gegenseitige Ausschluß zweier Antikörper bei der Bindung eines Dihapten-Konjugates. Das Konjugat besteht dabei aus dem Analyten und einem Fluorophor. Die beiden um die Konjugatbindung konkurrierenden Antikörper sind ein anti-Analyt Antikörper und ein anti-Fluorophor Antikörper, der zudem über die Eigenschaft verfügt, bei Bindung des Fluorophors dessen Fluoreszenz zu löschen. Externe Gaben des freien Analyten verschieben das eingestellte Gleichgewicht in Richtung Fluorophor-Bindung und damit Fluoreszenz-Löschung. Die Änderung der Fluoreszenz ist direkt an die Konzentration des freien Analyten gekoppelt und dient zu deren Bestimmung. Ein solcher Fluorescence Quenching Immunoassays wurde für die Konzentrationsbestimmung des Herbizides Diuron etabliert. Die erreichten Sensitivitäten erlauben die praktische, immundiagnostische Anwendung des Systems. Ein Dihapten-Konjugat wurde ebenfalls zum Aufbau eines Verfahrens zur Selektion Antikörper produzierender Zellen eingesetzt. Die Selektion der Antikörper produzierenden Zellen erfolgt unter Verwendung eines Toxinkonjugates. Dieses Konjugat besteht aus einem Liganden und einem Toxin. Die Antikörperbindung des Liganden behindert sterisch die Wechselwirkung der Toxinkomponente im Konjugat mit deren Zielstruktur in oder auf der Zelle. Nur Zellen die einen geeigneten Antikörper sezernieren, überleben die Selektion und reichern sich in der Kultur an. Das Selektionsverfahren wurde erfolgreich für die Selektion von E.coli Zellen eingesetzt, die einen rekombinanten, Fluorescein bindenden Antikörper produzierten. Das hierfür synthetisierte Toxinkonjugat bestand aus Fluorescein (Ligand) und Ampicillin (Toxinkomponente). Eine Ablösung der bisher für diese Aufgabe gebräuchlichen, außerordentlich kostenintensiven, Screening Methoden wird damit möglich. N2 - Homogeneous immunoassays are test systems which do not depend on separation steps. The substrate channeling immunoassay is based on the product/substrate transfer in an immunological complex built up by two enzymes. The product of the first enzyme functions as substrate for the second enzyme. The second enzyme generates a photometrically detectable product. The close proximity of these two enzymes is the basis of the substrate channeling. This proximity is created by antibody binding to the corresponding analyte. The enzymes glucose oxidase and peroxidase were used for the development of such an assay system. The established homogeneous immunoassay was functional. But the sensitivity of the assay was much lower than that of conventional heterogeneous immunoassays. The principle of a fluorescence quenching immunoassay is based on the fact that two antibodies exclude each other from binding to a dihapten conjugate. The conjugate consists of the analyte and the fluorophore. The two antibodies which compete for the conjugate binding are an anti-analyte antibody and an anti-fluorophore antibody. This anti-fluorophore antibody quenches the fluorescence of the fluorophore after binding. The addition of free analyte alters the equilibrium of the system so that the anti-fluorophore antibody is bound to the fluorophore and the fluorescence is quenched. The change in fluorescence is therefore an indicator of the concentration of free analyte added. A homogeneous fluorescence quenching immunoassay was established for the determination of the herbicide diuron. The sensitivities obtained allow the practical immunodiagnostic application of the system. A dihapten conjugate was also employed for the development of a selection method for antibody-producing cells. Toxin conjugates were used in this system. Each conjugate consisted of a ligand and a toxin. Antibody binding to the ligand sterically inhibits the toxin component to interact with its target structure. Only cells secreting a binding antibody will survive the selection and will accumulate in culture. The system was applied to the selection of E.coli cells producing a recombinant fluorescein-binding antibody. The toxin conjugate used in experiment consisted of fluorescein (ligand) and ampicillin (toxin component). This selection procedure allowed the isolation of recombinant antibody-producing E.coli cells. It has the potential to replace the time-consuming and labour-intensive methods used so far. KW - Homoger Immunoassay KW - Substate Channeling Immunoassay KW - Fluorescence Quenching Immunoassay KW - Selektion Antikörper-produzierender Zellen KW - Homogeneous immunoassay KW - Substrate channeling immunoassay KW - Fluorescence quenching immunoassay KW - Selection of antibody producing cells Y1 - 2007 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus-15982 ER - TY - JOUR A1 - Sellrie, Frank A1 - Warsinke, Axel A1 - Micheel, Burkhard T1 - Homogeneous indirect fluorescence quenching immunoassay for the determination of low molecular weight substances JF - Analytical & bioanalytical chemistry N2 - This paper describes the principle of a homogeneous indirect fluorescence quenching immunoassay that uses monoclonal antibodies. It is a carrier-free assay system that is performed completely in solution. The assay system was established for the determination of a low molecular weight substance (hapten), the herbicide diuron, used as a model analyte. A fluorescein-monuron conjugate together with a fluorescence-quenching monoclonal anti-fluorescein antibody and an anti-analyte antibody (here an anti-diuron/monuron monoclonal antibody) were used as central components of the assay. The fluorescein-monuron conjugate can be bound either by the anti-fluorescein monoclonal antibody or by the anti-diuron/ monuron monoclonal antibody. Due to steric hindrance, binding of both antibodies to the conjugate was not possible at the same time. By selecting the antibody concentrations appropriately, a dynamic equilibrium can be established that permits the preferential binding of the anti-diuron/monuron antibody to the conjugate, which allows the fluorescein in the conjugate to fluoresce. This equilibrium can be easily altered by adding free analyte (diuron), which competes with the conjugate to bind to the anti-diuron/monuron antibody. A reduction of anti-diuron/monuron antibody binding to the conjugate results in an increase in the binding of the anti-fluorescein antibody, which leads to a decrease in the fluorescence of the conjugate. The fluorescence is therefore a direct indicator of the state of equilibrium of the system and thus also the presence of free unconjugated analyte. The determination of an analyte based on this test principle does not require any washing steps. After the test components are mixed, the dynamic equilibrium is rapidly reached and the results can be obtained in less than 5 min by measuring the fluorescence of the fluorescein. We used this test principle for the determination of diuron, which was demonstrated for concentrations of approximately 5 nM. Y1 - 2008 UR - http://www.springerlink.com/content/n7227875454216v7/ VL - 386 IS - 2 SP - 206 EP - 210 ER - TY - JOUR A1 - Schenk, Jörg A. A1 - Sellrie, Frank A1 - Böttger, Volker A1 - Micheel, Burkhard A1 - Stöcklein, Walter F. M. T1 - Generation and application of a fluorescein-specific single chain antibody N2 - A recombinant single chain antibody fragment (designated scDE1) of the murine monoclonal anti-fluorescein antibody B13-DE1 was generated using the original hybridoma cells as source for the variable antibody heavy and light chain (VH and VL) genes. After cloning the variable genes into a phage vector a functional antibody fragment was selected by phage display panning. Recombinant antibody could be expressed as phage antibody and as soluble single chain antibody in Escherichia coli. High yield of scDE1 could also be detected in bacterial culture supernatant. The scDE1 showed the same binding specificity as the parental monoclonal antibody, i.e. it bound fluorescein, fluorescein derivatives and a fluorescein peptide mimotope. Surface plasmon resonance revealed a K(D) of 19 nM for the scDE1 compared to 0.7 nM for the monoclonal antibody. The isolated soluble scDE1 could easily be conjugated to horseradish peroxidase which allowed the use of the conjugate as universal indicator for the detection of fluorescein-labelled proteins in different immunoassays. Detection of hCG in urine was performed as a model system using scDE1. In addition to E. coli the scFv genes could also be transferred and expressed in eukaryotic cells. Finally, we generated HEK293 cells expressing the scDE1 at the cell surface. Y1 - 2007 UR - http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6VRJ-4P3DY33- 1&_user=1584062&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000053886&_version=1&_urlVersion=0&_userid=1584062&md5=e 4 ER - TY - JOUR A1 - Eisold, Ursula A1 - Sellrie, Frank A1 - Memczak, Henry A1 - Andersson, Anika A1 - Schenk, Jörg A. A1 - Kumke, Michael Uwe T1 - Dye tool box for a fluorescence enhancement immunoassay JF - Bioconjugate chemistry N2 - Immunochemical analytical methods are very successful in clinical diagnostics and are nowadays also emerging in the control of food as well as monitoring of environmental issues. Among the different immunoassays, luminescence based formats are characterized by their outstanding sensitivity making this format especially attractive for future applications. The need for multiparameter detection capabilities calls for a tool box of dye labels in order to transduce the biochemical reaction into an optically detectable signal. Here, in a multiparameter approach each analyte may be detected by a different dye with a unique emission color (covering the blue to red spectral range) or a unique luminescence decay kinetics. In the case of a competitive immunoassay format for each of the different dye labels an individual antibody would be needed. In the present paper a slightly modified approach is presented using a 7-aminocoumarin unit as the basic antigen against which highly specific antibodies were generated. Leaving the epitope region in the dyes unchanged but introducing a side group in positon 3 of the coumarin system allowed us to tune the optical properties of the coumarin dyes without the necessity of new antibody generation. Upon modification of the parent coumarin unit the full spectral range from blue to deep red was accessed. In the manuscript the photophysical characterization of the coumarin derivatives and their corresponding immunocomplexes with two highly specific antibodies is presented. The coumarin dyes and their immunocomplexes were characterized by steady-state and time-resolved absorption as well as emission spectroscopy. Moreover, fluorescence depolarization measurements were carried out to complement the data stressing the different binding modes of the two antibodies. The binding modes were evaluated using the photophysics of 7-aminocoumarins and how it was affected in the respective immunocomplexes, namely, the formation of the intramolecular charge transfer (ICT) as well as the twisted intramolecular charge transfer (TICT). In contrast to other antibody-dye pairs reported a distinct fluorescence enhancement upon formation of the antibody-dye complex up to a factor of SO was found. Because of the easy emission color tuning by tailoring the coumarin substitution for the antigen binding in nonrelevant position 3 of the parent molecule, a dye tool box is on hand which can be used in the construction of competitive multiparameter fluorescence enhancement immunoassays (FenIA). Y1 - 2018 U6 - https://doi.org/10.1021/acs.bioconjchem.7b00731 SN - 1043-1802 VL - 29 IS - 1 SP - 203 EP - 214 PB - American Chemical Society CY - Washington ER - TY - JOUR A1 - Sellrie, Frank A1 - Schenk, Jörg A. A1 - Behrsing, Olaf A1 - Drechsel, Oliver A1 - Micheel, Burkhard T1 - Cloning and characterization of a single chain antibody to glucose oxidase from a murine hybridoma N2 - Glucose oxidase (GOD) is an oxidoreductase catalyzing the reaction of glucose and oxygen to peroxide and gluconolacton (EC 1.1.3.4.). GOD is a widely used enzyme in biotechnology. Therefore the production of monoclonal antibodies and antibody fragments to GOD are of interest in bioanalytics and even tumor therapy. We describe here the generation of a panel of monoclonal antibodies to native and heat inactivated GOD. One of the hybridomas, E13BC8, was used for cloning of a single chain antibody (scFv). This scFv was expressed in Escherichia coli XL1-blue with the help of the vector system pOPE101. The scFv was isolated from the periplasmic fraction and detected by western blotting. It reacts specifically with soluble active GOD but does not recognize denatured GOD adsorbed to the solid phase. The same binding properties were also found for the monoclonal antibody E13BC8. Y1 - 2007 UR - http://www.jbmb.or.kr/fulltext/jbmb/view.php?vol=40&page=875 ER - TY - JOUR A1 - Sellrie, Frank A1 - Beck, Michael A1 - Hildebrandt, Niko A1 - Micheel, Burkhard T1 - A homogeneous time-resolved fluoroimmunoassay (TR-FIA) using antibody mediated luminescence quenching N2 - The determination of low-molecular weight substances (haptens) is demonstrated with a homogeneous time-resolved immunoassay using antibody-induced luminescence quenching. Our novel assay technology uses the newly developed monoclonal antibody (G24-BA9) to quench the luminescence of europium trisbipyridine (EuTBP). We performed a competitive biotin immunoassay including an EuTBP-biotin conjugate, the anti-EuTBP antibody G24-BA9 and streptavidin as assay components. Steric hindrance allows only the binding of either G24-BA9 (to the EuTBP moiety) or streptavidin (to the biotin moiety) to the EuTBP-biotin conjugate. Addition of the analyte biotin resulted in the binding of streptavidin to biotin and a concomitant preferred binding of G24-BA9 to EuTBP-biotin. Since G24-BA9 quenches the luminescence of EuTBP within the conjugate, the luminescence signal could be used to indicate and quantify the presence of free biotin in the system. All experiments were carried out in solution in the presence of 5% serum demonstrating the possibility of using our novel assay for a very fast determination of low molecular weight substances in biological fluids. Y1 - 2010 UR - http://www.rsc.org/Publishing/Journals/AY/Index.asp U6 - https://doi.org/10.1039/C0ay00306a SN - 1759-9660 ER - TY - JOUR A1 - Sellrie, Frank A1 - Schenk, Jörg A. A1 - Behrsing, Olaf A1 - Bottger, Volker A1 - Micheel, Burkhard T1 - A competitive immunoassay to detect a hapten using an enzyme-labelled peptide mimotope as tracer N2 - Mimotope peptides-peptides which mimic the binding of a hapten to its corresponding monoclonal antibody-were conjugated to peroxidase and used in competitive immunoassay. The established immunoassay was used to quantitatively determine the concentration of hapten. As model system in all the experiments described here, we used the binding of the monoclonal antibody B13-DE1 to fluorescein and the corresponding peptide mimotope. Y1 - 2002 UR - http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T2Y-44MGNDF- 1&_coverDate=03%2F01%2F2002&_alid=268992656&_rdoc=1&_fmt=&_orig=search&_qd=1&_cdi=4931&_sort=d&view=c&_acct=C000053886&_v e ER -