@article{WernerBehrsingScharteetal.2002, author = {Werner, Deljana and Behrsing, Olaf and Scharte, Gudrun and Woller, Jochen and Steup, Martin and Micheel, Burkhard}, title = {Monoclonal anti-diuron antibodies prevent inhibition of photosynthesis by diuron}, year = {2002}, language = {en} } @article{WarsinkeLettauWerneretal.2003, author = {Warsinke, Axel and Lettau, Kristian and Werner, Deljana and Micheel, Burkhard and Kwak, Young-Keun}, title = {Biomimetic Binders and Catalysts for Sensorics = Biomimetische Binder und Katalysatoren f{\"u}r die Sensorik}, year = {2003}, abstract = {Biosensors which make use of the high specificity of enzymes, antibodies, and nucleic acids have been described for detection of numerous metabolites, hormones, and nucleic acid sequences. In addition to biological components nowadays biomimetic recognition molecules are also used. Especially antibodies, aptamers, and molecular imprints are promising biomimetics. They could broaden the range of detectable analytes and could increase the functional stability of the sensor. In this publication we describe the generation of biomimetic antibodies and biomimetic molecular imprints for binding creatinine and for hydrolyzing phenylcarbamates to be used in electrochemical sensors.}, language = {de} } @article{WarnckeVogtUlrichetal.2004, author = {Warncke, Max and Vogt, Birgit and Ulrich, Jacqueline and von Laer, Meike Dorothee and Beyer, Winfried and Klump, Hannes and Micheel, Burkhard and Sheriff, Ahmed}, title = {Efficient in vitro transduction of naive murine B cells with lentiviral vectors}, year = {2004}, abstract = {The aim of this study was to determine the impact of lentiviral transduction on primary murine B cells. Studying B cell activities in vivo or using them for tolerance induction requires that the cells remain unaltered in their biological behavior except for expression of the transgene. As we show here, murine B cells can efficiently be transduced by lentiviral, VSV-G-pseudotyped vectors without the necessity of prior activation. Culture with LPS gave enhanced transduction efficiencies but led to the upregulation of CD86 and proliferation of the cells. Transduction of naive B cells by lentiviral vectors was dependent on multiplicity of infection and did not lead to a concomitant activation. Furthermore, the transduced cells could be used for studies in the NOD mouse system without altering the onset of diabetes. We conclude that lentiviral gene transfer into naive B cells is a powerful tool for manipulation of B cells for therapeutic applications.}, language = {en} } @article{WandHolzloehnerNeupertetal.2011, author = {Wand, Inga and Holzl{\"o}hner, Pamela and Neupert, Steffi and Micheel, Burkhard and Heilmann, Katja}, title = {Cooperation of dendritic cells with naive lymphocyte populations to induce the generation of antigen-specific antibodies in vitro}, series = {Journal of biotechnology}, volume = {156}, journal = {Journal of biotechnology}, number = {3}, publisher = {Elsevier}, address = {Amsterdam}, issn = {0168-1656}, doi = {10.1016/j.jbiotec.2011.09.002}, pages = {173 -- 181}, year = {2011}, abstract = {The production of monoclonal antibodies by hybridoma technology is dependent on lymphocytes taken from vertebrates which have to be immunized against the corresponding antigen. We present here our first experiments which should allow the replacement of this in vivo immunization step by an in vitro immunization procedure. This work provides new possibilities for the specific activation of immune cells in order to use them for the generation of antibodies which are not of murine origin. Bone marrow-derived dendritic cells were loaded with antigen and co-cultured with naive T and B lymphocytes of non-immunized mice. The interaction and activation of the different cell types were investigated by measuring the expression of specific cell surface markers, the release of activation-dependent interleukins and the secretion of antigen-specific antibodies. We could demonstrate that dendritic cells process and present antigen fragments and activate T cells, that T cells proliferate and release activation-induced interleukins, and that B cells maturate under the influence of activated T cells and secrete antigen-specific antibodies.}, language = {en} } @article{VogtWarnckeMicheeletal.2009, author = {Vogt, Birgit and Warncke, Marit and Micheel, Burkhard and Sheriff, Ahmed}, title = {Lentiviral gene transfer of CTLA4 generates B cells with reduced costimulatory properties : brief definite report}, issn = {0891-6934}, doi = {10.1080/08916930902832470}, year = {2009}, abstract = {Peripheral T-cell (TC) tolerance can be induced by tolerogenic antigen-presenting cell (APC). A prerequisite is the reduction or blockade of B7 of APC. Besides dendritic cell, B cells can be used as APC. Here, we show the generation B cells with reduced B7 expression by lentiviral transduction of endoplasmic reticulum (ER)-directed CTLA4. Vectors coding for the human CTL4-Ig were used for the human B-cell line Raji. Transduction efficiency was over 90\% (MOI = 3). For the murine B-cell line A20 and for primary mouse B cells, murine CTLA4 was used. We show that B cells with reduced B7 expression reduce the antigen (Ag) specific TC proliferation in vitro. B cells expressing an ER-directed CTLA4 may reduce Ag-specific immune responses.}, language = {en} } @article{VanderVenEhlerVakeeletal.2006, author = {VanderVen, Peter F. M. and Ehler, Elisabeth and Vakeel, Padmanabhan and Eulitz, Stefan and Schenk, J{\"o}rg A. and Milting, Hendrik and Micheel, Burkhard and F{\"u}rst, Dieter Oswald}, title = {Unusual splicing events result in distinct Xin isoforms that associate differentially with filamin c and Mena/ VASP}, doi = {10.1016/j.yexcr.2006.03.015}, year = {2006}, abstract = {Filamin c is the predominantly expressed filamin isoform in striated muscles. It is localized in myofibrillar Z- discs, where it binds FATZ and myotilin, and in myotendinous junctions and intercalated discs. Here, we identify Xin, the protein encoded by the human gene 'cardiomyopathy associated 1' (CMYA1) as filamin c binding partner at these specialized structures where the ends of myofibrils are attached to the sarcolemma. Xin directly binds the EVH1 domain proteins Mena and VASP. In the adult heart, Xin and Mena/VASP colocalize with filamin c in intercalated discs. In cultured cardiomyocytes, the proteins also localize in the nonstriated part of myofibrils, where sarcomeres are assembled and an extensive reorganization of the actin cytoskeleton occurs. Unusual intraexonic splicing events result in the existence of three Xin isoforms that associate differentially with its ligands. The identification of the complex filamin c-Xin-Mena/VASP provides a first glance on the role of Xin in the molecular mechanisms involved in developmental and adaptive remodeling of the actin cytoskeleton during cardiac morphogenesis and sarcomere assembly. (c) 2006 Elsevier Inc. All rights reserved}, language = {en} } @article{StoeckleinWarsinkeMicheeletal.1998, author = {St{\"o}cklein, Walter F. M. and Warsinke, Axel and Micheel, Burkhard and Kempter, Gerhard and H{\"o}hne, Wolfgang and Scheller, Frieder W.}, title = {Diphenylurea hapten sensing with a monoclonal antibody and its Fab fragment : kinetic and thermodynamic investigations}, year = {1998}, language = {en} } @article{StoeckleinWarsinkeMicheeletal.1998, author = {St{\"o}cklein, Walter F. M. and Warsinke, Axel and Micheel, Burkhard and H{\"o}hne, Wolfgang and Woller, Jochen and Kempter, Gerhard and Scheller, Frieder W.}, title = {Characterization of a monoclonal antibody and its Fab fragment against diphenylurea hapten with BIA}, isbn = {3-8154-3540-4}, year = {1998}, language = {en} } @article{StoeckleinRohdeScharteetal.2000, author = {St{\"o}cklein, Walter F. M. and Rohde, M. and Scharte, Gudrun and Behrsing, Olaf and Warsinke, Axel and Micheel, Burkhard and Scheller, Frieder W.}, title = {Sensitive detection of triazine and phenylurea pesticides in pure organic solvent by enzyme linked immunsorbent assay (ELISA): stabilities, solubilities and sensitives}, year = {2000}, language = {en} } @article{StoeckleinBehrsingScharteetal.2000, author = {St{\"o}cklein, Walter F. M. and Behrsing, Olaf and Scharte, Gudrun and Micheel, Burkhard and Benkert, Alexander and Sch{\"o}ssler, W. and Warsinke, Axel and Scheller, Frieder W.}, title = {Enzyme kinetic assays with surface plasmon resonance (BIAcore) based on competition between enzyme and creatinine antibody}, year = {2000}, language = {en} } @article{StuckasMesserschmidtPutzleretal.2009, author = {Stuckas, Heiko and Messerschmidt, Katrin and Putzler, Sascha and Baumann, Otto and Schenk, J{\"o}rg A. and Tiedemann, Ralph and Micheel, Burkhard}, title = {Detection and characterization of gamete-specific molecules in Mytilus edulis using selective antibody production}, issn = {1040-452X}, doi = {10.1002/Mrd.20916}, year = {2009}, abstract = {The mussel Mytilus edulis can be used as model to study the molecular basis of reproductive isolation because this species maintains its species integrity, despite of hybridizing in zones of contact with the closely related species M. trossulus or M. galloprovincialis. This study uses selective antibody production by means of hybridoma technology to identify molecules which are involved in sperm function of M. edulis. Fragmented sperm were injected into mice and 25 hybridoma cell clones were established to obtain monoclonal antibodies (mAb). Five clones were identified producing mAb targeting molecules putatively involved in sperm function based on enzyme immunoassays, dot and Western blotting as well as immunostaining of tissue sections. Specific localization of these mAb targets on sperm and partly also in somatic tissue suggests that all five antibodies bind to different molecules. The targets of the mAb obtained from clone G26-AG8 were identified using mass spectrometry (nano-LC-ESI-MS/MS) as M6 and M7 lysin. These acrosomal proteins have egg vitelline lyses function and are highly similar (76\%) which explains the cross reactivity of mAb G26- AG8. Furthermore, M7 lysin was recently shown to be under strong positive selection suggesting a role in interspecific reproductive isolation. This study shows that M6 and M7 lysin are not only found in the sperm acrosome but also in male somatic tissue of the mantle and the posterior adductor muscle, while being completely absent in females. The monoclonal antibody G26-AG8 described here will allow elucidating M7/M6 lysin function in somatic and gonad tissue of adult and developing animals.}, language = {en} } @article{StechMerkSchenketal.2012, author = {Stech, Marlitt and Merk, Helmut and Schenk, J{\"o}rg A. and St{\"o}cklein, Walter F. M. and W{\"u}stenhagen, Doreen Anja and Micheel, Burkhard and Duschl, Claus and Bier, Frank Fabian and Kubick, Stefan}, title = {Production of functional antibody fragments in a vesicle-based eukaryotic cell-free translation system}, series = {Journal of biotechnology}, volume = {164}, journal = {Journal of biotechnology}, number = {2}, publisher = {Elsevier}, address = {Amsterdam}, issn = {0168-1656}, doi = {10.1016/j.jbiotec.2012.08.020}, pages = {220 -- 231}, year = {2012}, abstract = {Cell-free protein synthesis is of increasing interest for the rapid and high-throughput synthesis of many proteins, in particular also antibody fragments. In this study, we present a novel strategy for the production of single chain antibody fragments (scFv) in a eukaryotic in vitro translation system. This strategy comprises the cell-free expression, isolation and label-free interaction analysis of a model antibody fragment synthesized in two differently prepared insect cell lysates. These lysates contain translocationally active microsomal structures derived from the endoplasmic reticulum (ER), allowing for posttranslational modifications of cell-free synthesized proteins. Both types of these insect cell lysates enable the synthesis and translocation of scFv into ER-derived vesicles. However, only the one that has a specifically adapted redox potential yields functional active antibody fragments. We have developed a new methodology for the isolation of functional target proteins based on the translocation of cell-free produced scFv into microsomal structures and subsequent collection of protein-enriched vesicles. Antibody fragments that have been released from these vesicles are shown to be well suited for label-free binding studies. Altogether, these results show the potential of insect cell lysates for the production, purification and selection of antibody fragments in an easy-to-handle and time-saving manner.}, language = {en} } @article{SheriffVogtBaumgartetal.2003, author = {Sheriff, Ahmed and Vogt, B. and Baumgart, Martin and Montag, C. and Hollenbach, B. and Schenk, J{\"o}rg A. and Ulrich, J. and Ellias, F. and Micheel, Burkhard}, title = {Intracellular capture of B7 in antigen-presenting cells reduces costimulatory activity}, year = {2003}, abstract = {CTLA-4 gene constructs were designed to express CTLA-4 exclusively in the endoplasmic reticulum (ER). Four different CTLA-4 gene constructs were transfected into HEK 293 (human embryonic kidney) and A20 (Balb/c mouse B lymphoma) cells. All constructs contained an ER retention signal and coded for CTLA-4 expression in the ER. One of the constructs, which contained the membrane part of CTLA-4, coded for an expression both on the cell surface and in the ER. Three of the expressed CTLA-4 types (including the ER-membrane-expressed form) caused a reduced surface expression of B7 in the A20 cells. Only constructs which allow dimerization of CTLA-4 showed this effect. It is assumed that intracellular CTLA-4 bound B7 and inhibited therefore the transport of B7 to the surface. The binding obviously caused also an enhanced degradation of the complexes because both proteins showed a low concentration in the transfected cell lines. CTLA-4-transfected and B7-reduced A20 cells showed a diminished costimulating activity upon T cells. This was demonstrated by a reduced proliferation of T cells from ovalbumin-immunized Balb/c mice, incubated with ovalbumin peptide-primed CTLA-4-transfected A20 cells.}, language = {en} } @article{SellrieWarsinkeMicheel2008, author = {Sellrie, Frank and Warsinke, Axel and Micheel, Burkhard}, title = {Homogeneous indirect fluorescence quenching immunoassay for the determination of low molecular weight substances}, series = {Analytical \& bioanalytical chemistry}, volume = {386}, journal = {Analytical \& bioanalytical chemistry}, number = {2}, pages = {206 -- 210}, year = {2008}, abstract = {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.}, language = {en} } @article{SellrieSchenkBehrsingetal.2007, author = {Sellrie, Frank and Schenk, J{\"o}rg A. and Behrsing, Olaf and Drechsel, Oliver and Micheel, Burkhard}, title = {Cloning and characterization of a single chain antibody to glucose oxidase from a murine hybridoma}, year = {2007}, abstract = {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.}, language = {en} } @article{SellrieSchenkBehrsingetal.2002, author = {Sellrie, Frank and Schenk, J{\"o}rg A. and Behrsing, Olaf and Bottger, Volker and Micheel, Burkhard}, title = {A competitive immunoassay to detect a hapten using an enzyme-labelled peptide mimotope as tracer}, year = {2002}, abstract = {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.}, language = {en} } @article{SellrieBeckHildebrandtetal.2010, author = {Sellrie, Frank and Beck, Michael and Hildebrandt, Niko and Micheel, Burkhard}, title = {A homogeneous time-resolved fluoroimmunoassay (TR-FIA) using antibody mediated luminescence quenching}, issn = {1759-9660}, doi = {10.1039/C0ay00306a}, year = {2010}, abstract = {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.}, language = {en} } @article{SchloerHolzloehnerListeketal.2018, author = {Schl{\"o}r, Anja and Holzl{\"o}hner, Pamela and Listek, Martin and Grieß, Cindy and Butze, Monique and Micheel, Burkhard and Hentschel, Christian and Sowa, Mandy and Roggenbuck, Dirk and Schierack, Peter and F{\"u}ner, Jonas and Schliebs, Erik and Goihl, Alexander and Reinhold, Dirk and Hanack, Katja}, title = {Generation and validation of murine monoclonal and camelid recombinant single domain antibodies specific for human pancreatic glycoprotein 2}, series = {New biotechnology}, volume = {45}, journal = {New biotechnology}, publisher = {Elsevier}, address = {Amsterdam}, issn = {1871-6784}, doi = {10.1016/j.nbt.2018.03.006}, pages = {60 -- 68}, year = {2018}, abstract = {Pancreatic secretory zymogen-granule membrane glycoprotein 2 (GP2) has been identified as a major autoantigenic target in Crohn's disease patients. It was reported recently that a long (GP2a) and a short (GP2b) isoform of GP2 exist and that in the outcome of inflammatory bowel diseases (IBD) GP2-specific autoantibodies probably appear as new serological markers for diagnosis and therapeutic monitoring. To investigate this further and in order to establish diagnostic tools for the discrimination of both GP2 isoforms, a set of different murine monoclonal and camelid recombinant single domain antibodies (camelid VHH) was generated and validated in various enzyme-linked immunosorbent assay (ELISA) formats, immunofluorescence on transgenic cell lines and immunohistochemistry on monkey pancreas tissue sections. Out of six binders identified, one was validated as highly specific for GP2a. This murine monoclonal antibody (mAb) was used as capture antibody in construction of a sandwich ELISA for the detection of GP2a. Camelid VHHs or a second murine mAb served as detection antibodies in this system. All antibodies were also able to stain GP2a or GP2b on transgenic cell lines as well as on pancreatic tissue in immunohistochemistry. The KD values measured for the camelid VHHs were between 7 nM and 23pM. This set of specific binders will enable the development of suitable diagnostic tools for GP2-related studies in IBD.}, language = {en} } @article{SchlagOsterzielOezceliketal.2008, author = {Schlag, Peter M. and Osterziel, Karl Joseph and {\"O}zcelik, Cemil and Scherneck, Siegfried and Wenzel, Katrin and Daskalow, Katjana and Herse, Florian and Seitz, Susanne and Zacharias, Ute and Schenk, J{\"o}rg A. and Schulz, Herbert and H{\"u}bner, Norbert and Micheel, Burkhard}, title = {The protein phosphatase 1 inhibitor KEPI is down regulated in breast cancer cell lines and tissues and involved in the regulation of the tumour suppressor EGR1 via the MEK-ERK pathway}, year = {2008}, abstract = {KEPI is a protein kinase C-potentiated inhibitory protein for type 1 Ser/Thr protein phosphatases. We found no or reduced expression of KEPI in breast cancer cell lines, breast tumors and metastases in comparison to normal breast cell lines and tissues, respectively. KEPI protein expression and ubiquitous localization was detected with a newly generated antibody. Ectopic KEPI expression in MCF7 breast cancer cells induced differential expression of 95 genes, including the up-regulation of the tumor suppressors EGR1 (early growth response 1) and PTEN (phosphatase and tensin homolog), which is regulated by EGR1. We further show that the up-regulation of EGR1 in MCF7/KEPI cells is mediated by MEK-ERK signaling. The inhibition of this pathway by the MEK inhibitor UO126 led to a strong decrease in EGR1 expression in MCF7/KEPI cells. These results reveal a novel role for KEPI in the regulation of the tumor suppressor gene EGR1 via activation of the MEK-ERK MAPK pathway.}, language = {en} } @article{SchenkSellrieBoettgeretal.2007, author = {Schenk, J{\"o}rg A. and Sellrie, Frank and B{\"o}ttger, Volker and Micheel, Burkhard and St{\"o}cklein, Walter F. M.}, title = {Generation and application of a fluorescein-specific single chain antibody}, year = {2007}, abstract = {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.}, language = {en} }