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 - TY - JOUR A1 - Ettlinger, Julia A1 - Schenk, Jörg A. A1 - Micheel, Burkhard A1 - Ehrentreich-Förster, Eva A1 - Gajovic-Eichelmann, Nenad T1 - A direct competitive homogeneous immunoassay for progesterone - the Redox Quenching Immunoassay JF - Electroanalysis : an international journal devoted to fundamental and practical aspects of electroanalysis N2 - A direct competitive amperometric immunoassay format for the detection of haptens and proteins was developed. The method is based on the quenching of electroactivity of ferrocenium, which is coupled to the antigen and used as the primary reporter, upon binding to a monoclonal anti-ferrocenium antibody, which is coupled to the detection antibody and used as a secondary reporter. A separation-free progesterone immunoassay with a lower detection limit of 1 ng?mL-1 (3.18 nmol?L-1) in 1?:?2 diluted blood serum was realised by combining two bifunctional conjugates, a ferrocenium-PEG-progesterone tracer and a bioconjugate of one anti-progesterone and one anti-ferrocenium antibody. The immune complex is formed within 30 s upon addition of progesterone, resulting in a total analysis time of 1.5 min. KW - Immunoassay KW - Amperometry KW - Ferrocene KW - Progesterone Y1 - 2012 U6 - https://doi.org/10.1002/elan.201200107 SN - 1040-0397 VL - 24 IS - 7 SP - 1567 EP - 1575 PB - Wiley-VCH CY - Weinheim ER - TY - JOUR A1 - Bergholz, André A1 - Heymann, Stephan A1 - Schenk, Jörg A. A1 - Freytag, Johann Christoph T1 - Biological sequences integrated: a relational database approach N2 - Over the last decade the modeling and the storage of biological data has been a topic of wide interest for scientists dealing with biological and biomedical research. Currently most data is still stored in text files which leads to data redundancies and file chaos. In this paper we show how to use relational modeling techniques and relational database technology for modeling and storing biological sequence data, i.e. for data maintained in collections like EMBL or SWISS-PROT to better serve the needs for these application domains. For this reason we propose a two step approach. First, we model the structure (and therefore the meaning of the) data using an Entity-Relationship approach. The ER model leads to a clean design of a relational database schema for storing and retrieving the DNA and protein data extracted from various sources. Our approach provides the clean basis for building complex biological applications that are more amenable to changes and software ports than their file-base counterparts. Y1 - 2001 UR - http://www.springerlink.com/app/home/ contribution.asp?wasp=161c4c19086a4dceac9312b46e5f2348&referrer=parent&backto=issue,1,5;journal,14,29;linkingpublicationr esults,1:102835,1 ER - TY - JOUR A1 - Lawatscheck, Robert A1 - Aleksaite, Egle A1 - Schenk, Jörg A. A1 - Micheel, Burkhard A1 - Jandrig, Burkhard A1 - Holland, Gudrun A1 - Sasnauskas, Kestutius A1 - Gedvilaite, Alma A1 - Ulrich, Rainer Günter T1 - Chimeric polyomavirus-derived virus-like particles : the immunogenicity of an inserted peptide applied without adjuvant to mice depends on its insertion site and its flanking linker sequence N2 - We inserted the sequence of the carcinoembryonic antigen-derived T cell epitope CAP-1-6D (CEA) into different positions of the hamster polyomavirus major capsid protein VP1. Independently from additional flanking linkers, yeast- expressed VP1 proteins harboring the CEA insertion between VP1 amino acid residues 80 and 89 (site 1) or 288 and 295 (site 4) or simultaneously at both positions assembled to chimeric virus-like particles (VLPs). BALB/c mice immunized with adjuvant-free VLPs developed VP1- and epitope-specific antibodies. The level of the CEA-specific antibody response was determined by the insertion site, the number of inserts, and the flanking linker. The strongest CEA-specific antibody response was observed in mice immunized with VP1 proteins harboring the CEA insert at site 1. Moreover, the CEA- specific antibodies in these mice were still detectable 6 mo after the final booster immunization. Our results indicate that hamster polyomavirus-derived VLPs represent a highly immunogenic carrier for foreign insertions that might be useful for clinical and therapeutic applications. Y1 - 2007 UR - http://www.liebertonline.com/vim U6 - https://doi.org/10.1089/vim.2007.0023 SN - 0882-8245 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 - Kuhne, Maren A1 - Dippong, Martin A1 - Flemig, Sabine A1 - Hoffmann, Katrin A1 - Petsch, Kristin A1 - Schenk, Jörg A. A1 - Kunte, Hans-Jörg A1 - Schneider, Rudolf J. T1 - Comparative characterization of mAb producing hapten-specific hybridoma cells by flow cytometric analysis and ELISA JF - Journal of immunological methods N2 - A novel method that optimizes the screening for antibody-secreting hapten-specific hybridoma cells by using flow cytometry is described. Cell clones specific for five different haptens were analyzed. We selectively double stained and analyzed fixed hybridoma cells with fluorophore-labeled haptens to demonstrate the target-selectivity, and with a fluorophore-labeled anti-mouse IgG antibody to characterize the level of surface expression of membrane-bound IgGs. ELISA measurements with the supernatants of the individual hybridoma clones revealed that antibodies from those cells, which showed the highest fluorescence intensities in the flow cytometric analysis, also displayed the highest affinities for the target antigens. The fluorescence intensity of antibody-producing cells corresponded well with the produced antibodies' affinities toward their respective antigens. Immunohistochemical staining verified the successful double labeling of the cells. Our method makes it possible to perform a high-throughput screening for hybridoma cells, which have both an adequate IgG production rate and a high target affinity. (C) 2014 Elsevier B.V. All rights reserved. KW - Immunization KW - Hapten KW - Monoclonal antibodies KW - Hybridoma KW - Flow cytometry KW - ELISA Y1 - 2014 U6 - https://doi.org/10.1016/j.jim.2014.07.004 SN - 0022-1759 SN - 1872-7905 VL - 413 SP - 45 EP - 56 PB - Elsevier CY - Amsterdam ER - TY - JOUR A1 - Stuckas, Heiko A1 - Messerschmidt, Katrin A1 - Putzler, Sascha A1 - Baumann, Otto A1 - Schenk, Jörg A. A1 - Tiedemann, Ralph A1 - Micheel, Burkhard T1 - Detection and characterization of gamete-specific molecules in Mytilus edulis using selective antibody production N2 - 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. Y1 - 2009 UR - http://www3.interscience.wiley.com/cgi-bin/jhome/37692 U6 - https://doi.org/10.1002/Mrd.20916 SN - 1040-452X 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 - Pecher, Gabriele A1 - Schirrmann, Thomas A1 - Kaiser, Lothar A1 - Schenk, Jörg A. T1 - Efficient cryopreservation of dendritic cells transfected with cDNA of a tumour antigen for clinical application N2 - Dendritic cells (DCs) are the most potent antigen-presenting cells of the immune system and are currently being investigated in clinical applications as cancer vaccines. An efficient cryopreservation method would greatly contribute to their use in clinical trials. We have established a method for freezing of DCs derived from peripheral blood mononuclear cells using the plasma expander Gelifundol. This enabled us to reduce the concentration of the toxic DMSO to 5%. The method could be performed without the addition of fetal calf serum or any other serum. After freezing, the viability of the DCs was 90%. The cells exhibited all the phenotypic characteristics (CD11c+, HLA-DR+, CD80+, CD83+, CD86+) of DCs, as tested by flow cytometry. Cells transfected with cDNA for the tumour antigen mucin expressed this protein on their surfaces in the same manner as before freezing. The stimulating capacity of a mixed lymphocyte culture was also preserved. These findings offer an efficient method for the cryopreservation of DCs for use in clinical trials. Y1 - 2001 UR - http://www.babonline.org/bab/034/0161/bab0340161.htm 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 -