@article{MichelchenMicheelHanack2021, author = {Michelchen, Sophia and Micheel, Burkhard and Hanack, Katja}, title = {In vitro immunization approach to generate specific murine monoclonal IgG antibodies}, series = {Journal of immunological methods : JIM}, volume = {499}, journal = {Journal of immunological methods : JIM}, publisher = {Elsevier}, address = {Amsterdam}, issn = {0022-1759}, doi = {10.1016/j.jim.2021.113149}, pages = {8}, year = {2021}, abstract = {Generating a monoclonal antibody to date is a time intense process that requires immunization of laboratory animals. The transfer of the humoral immune response into in vitro settings enables a shortening of this process and circumvents the necessity of in vivo immunization. However, to orchestrate the complex interplay of dendritic cells, T and B lymphocytes in vitro is very challenging. We therefore aimed for a simplified approach focusing on the protagonist of antibody production: the B lymphocyte. We activated purified murine B lymphocytes alone in vitro by using combinations of antigen and stimuli. We were able to induce a specific antibody response within ten days of culture against a viral coat protein as model antigen. Antibodies were of both IgM and IgG subclass. The stimulated B lymphocytes were transformed into permanently antibody-producing hybridomas by cell fusion technology. We furthermore used this method to induce a specific antibody response against L. pneumophila in vitro. We thus established a useful and effective in vitro protocol to generate monoclonal antibodies. By overcoming the necessity of in vivo immunization this protocol may be the first step towards a universal strategy to generate antibodies from various species.}, language = {en} } @article{EngelMicheelHanack2022, author = {Engel, Robert and Micheel, Burkhard and Hanack, Katja}, title = {Three-dimensional cell culture approach for in vitro immunization and the production of monoclonal antibodies}, series = {Biomedical materials : materials for tissue engineering and regenerative medicine}, volume = {17}, journal = {Biomedical materials : materials for tissue engineering and regenerative medicine}, number = {5}, publisher = {Inst. of Physics}, address = {London}, issn = {1748-6041}, doi = {10.1088/1748-605X/ac7b00}, pages = {11}, year = {2022}, abstract = {The generation of monoclonal antibodies using an in vitro immunization approach is a promising alternative to conventional hybridoma technology. As recently published, the in vitro approach enables an antigen-specific activation of B lymphocytes within 10-12 d followed by immortalization and subsequent selection of hybridomas. This in vitro process can be further improved by using a three-dimensional surrounding to stabilize the complex microenvironment required for a successful immune reaction. In this study, the suitability of Geltrex as a material for the generation of monoclonal antigen-specific antibodies by in vitro immunization was analyzed. We could show that dendritic cells, B cells, and T cells were able to travel through and interact inside of the matrix, leading to the antigen-specific activation of T and B cells. For cell recovery and subsequent hybridoma technique the suitability of dispase and Corning cell recovery solution (CRS) was compared. In our experiments, the use of dispase resulted in a severe alteration of cell surface receptor expression patterns and significantly higher cell death, while we could not detect an adverse effect of Corning CRS. Finally, an easy approach for high-density cell culture was established by printing an alginate ring inside a cell culture vessel. The ring was filled with Geltrex, cells, and medium to ensure a sufficient supply during cultivation. Using this approach, we were able to generate monoclonal hybridomas that produce antigen-specific antibodies against ovalbumin and the SARS-CoV-2 nucleocapsid protein.}, language = {en} } @article{HolzloehnerButzeMaieretal.2018, author = {Holzl{\"o}hner, Pamela and Butze, Monique and Maier, Natalia and Hebel, Nicole and Schliebs, Erik and Micheel, Burkhard and Fuener, Jonas and Heidicke, Gabriele and Hanack, Katja}, title = {Generation of murine monoclonal antibodies with specificity against conventional camelid IgG1 and heavy-chain only IgG2/3}, series = {Veterinary Immunology and Immunopathology}, volume = {197}, journal = {Veterinary Immunology and Immunopathology}, publisher = {Elsevier}, address = {Amsterdam}, issn = {0165-2427}, doi = {10.1016/j.vetimm.2018.01.006}, pages = {1 -- 6}, year = {2018}, abstract = {Camelids possess antibodies with a conventional four-chain structure consisting of two heavy and two light chains (of subclass IgG1) but further they also generate heavy-chain only antibodies (of subclass IgG2 and 3) which are fully functional in antigen binding. In this study subclass-specific murine monoclonal antibodies specific to conventional camelid IgG1 and heavy-chain only IgG2/3 were generated and validated for the use as potent secondary detection reagents. The monoclonal antibodies are able to differentiate between all camelid IgGs, conventional four-chain camelid antibodies (of subclass IgG1) and exclusively heavy chain-only antibodies (of subclasses IgG2 and IgG3). Further these antibodies were used to detect specific immune responses after vaccination of Camelids against bovine corona- and rotavirus strains and different E.coli. and Clostridia - antigens and to identify Erysipelothrix rhusiopathiae infected animals within a herd. The described antibodies are suitable as new secondary agents for the detection of different camelid subclasses and the validation of camelid immune reactions.}, 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{LuetkecosmannFaupelPorstmannetal.2019, author = {Luetkecosmann, Steffi and Faupel, Thomas and Porstmann, Silvia and Porstmann, Tomas and Micheel, Burkhard and Hanack, Katja}, title = {A cross-reactive monoclonal antibody as universal detection antibody in autoantibody diagnostic assays}, series = {Clinica chimica acta}, volume = {499}, journal = {Clinica chimica acta}, publisher = {Elsevier}, address = {Amsterdam}, issn = {0009-8981}, doi = {10.1016/j.cca.2019.09.003}, pages = {87 -- 92}, year = {2019}, abstract = {Diagnostics of Autoimmune Diseases involve screening of patient samples for containing autoantibodies against various antigens. To ensure quality of diagnostic assays a calibrator is needed in each assay system. Different calibrators as recombinant human monoclonal antibodies as well as chimeric antibodies against the autoantigens of interest are described. A less cost-intensive and also more representative possibility covering different targets on the antigens is the utilization of polyclonal sera from other species. Nevertheless, the detection of human autoantibodies as well as the calibration reagent containing antibodies from other species in one assay constitutes a challenge in terms of assay calibration. We therefore developed a cross-reactive monoclonal antibody which binds human as well as rabbit sera with similar affinities in the nanomolar range. We tested our monoclonal antibody S38CD11B12 successfully in the commercial Serazym (R) Anti-Cardiolipin-beta 2-GPI IgG/IgM assay and could thereby prove the eligibility of S38CD11B12 as detection antibody in autoimmune diagnostic assays using rabbit derived sera as reference material.}, language = {en} } @article{MesserschmidtDegenMicheel2011, author = {Messerschmidt, Katrin and Degen, Janine and Micheel, Burkhard}, title = {Oxidoreductase activity of multifunctional monoclonal antibody B13-DE1}, series = {Journal of molecular recognition : an international journal devoted to research on specific molecular recognition in chemistry, biology, biotechnology and medicine}, volume = {24}, journal = {Journal of molecular recognition : an international journal devoted to research on specific molecular recognition in chemistry, biology, biotechnology and medicine}, number = {6}, publisher = {Wiley-Blackwell}, address = {Malden}, issn = {0952-3499}, doi = {10.1002/jmr.1136}, pages = {930 -- 934}, year = {2011}, abstract = {The monoclonal antibody B13-DE1 binds fluorescein, several fluorescein derivatives, and three peptide mimotopes. Our results revealed that this antibody also catalyzed the redox reaction of resazurin to resorufin, which are both structurally related to fluorescein. By using sodium sulfite as a reducing agent, the antibody B13-DE1 lowered the activation energy of this reaction. The Michaelis-Menten constant and turnover number of the catalyzed reaction were determined as 4.2 mu mol/l and 0.0056 s(-1), respectively. Because the results showed that fluorescein inhibited the catalytic activity of the antibody, we assume that the antigen-binding site and the catalytic active site are identical.}, 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{EttlingerSchenkMicheeletal.2012, author = {Ettlinger, Julia and Schenk, J{\"o}rg A. and Micheel, Burkhard and Ehrentreich-F{\"o}rster, Eva and Gajovic-Eichelmann, Nenad}, title = {A direct competitive homogeneous immunoassay for progesterone - the Redox Quenching Immunoassay}, series = {Electroanalysis : an international journal devoted to fundamental and practical aspects of electroanalysis}, volume = {24}, journal = {Electroanalysis : an international journal devoted to fundamental and practical aspects of electroanalysis}, number = {7}, publisher = {Wiley-VCH}, address = {Weinheim}, issn = {1040-0397}, doi = {10.1002/elan.201200107}, pages = {1567 -- 1575}, year = {2012}, abstract = {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.}, language = {en} } @article{HessBartelRothetal.2012, author = {Hess, Anne-Katrin and Bartel, Manuela and Roth, Karina and Messerschmidt, Katrin and Heilmann, Katja and Kenchington, Ellen and Micheel, Burkhard and Stuckas, Heiko}, title = {Expression of M6 and M7 lysin in Mytilus edulis is not restricted to sperm, but occurs also in oocytes and somatic tissue of males and females}, series = {Molecular reproduction and development}, volume = {79}, journal = {Molecular reproduction and development}, number = {8}, publisher = {Wiley-Blackwell}, address = {Hoboken}, issn = {1040-452X}, doi = {10.1002/mrd.22056}, pages = {517 -- 524}, year = {2012}, abstract = {Sperm proteins of marine sessile invertebrates have been extensively studied to understand the molecular basis of reproductive isolation. Apart from molecules such as bindin of sea urchins or lysin of abalone species, the acrosomal protein M7 lysin of Mytilus edulis has been analyzed. M7 lysin was found to be under positive selection, but mechanisms driving the evolution of this protein are not fully understood. To explore functional aspects, this study investigated the protein expression pattern of M7 and M6 lysin in gametes and somatic tissue of male and female M. edulis. The study employs a previously published monoclonal antibody (G26-AG8) to investigate M6 and M7 lysin protein expression, and explores expression of both genes. It is shown that these proteins and their encoding genes are expressed in gametes and somatic tissue of both sexes. This is in contrast to sea urchin bindin and abalone lysin, in which gene expression is strictly limited to males. Although future studies need to clarify the functional importance of both acrosomal proteins in male and female somatic tissue, new insights into the evolution of sperm proteins in marine sessile invertebrates are possible. This is because proteins with male-specific expression (bindin, lysin) might evolve differently than proteins with expression in both sexes (M6/M7 lysin), and the putative function of both proteins in females opens the possibility that the evolution of M6/M7 lysin is under sexual antagonistic selection, for example, mutations beneficial to the acrosomal function that are less beneficial the function in somatic tissue of females.Mol. Reprod. Dev. 79: 517-524, 2012.}, language = {en} } @article{BartelHartmannLehmannetal.2012, author = {Bartel, Manuela and Hartmann, Stefanie and Lehmann, Karola and Postel, Kai and Quesada, Humberto and Philipp, Eva E. R. and Heilmann, Katja and Micheel, Burkhard and Stuckas, Heiko}, title = {Identification of sperm proteins as candidate biomarkers for the analysis of reproductive isolation in Mytilus: a case study for the enkurin locus}, series = {Marine biology : international journal on life in oceans and coastal waters}, volume = {159}, journal = {Marine biology : international journal on life in oceans and coastal waters}, number = {10}, publisher = {Springer}, address = {New York}, issn = {0025-3162}, doi = {10.1007/s00227-012-2005-7}, pages = {2195 -- 2207}, year = {2012}, abstract = {Sperm proteins of the marine sessile mussels of the Mytilus edulis species complex are models to investigate reproductive isolation and speciation. This study aimed at identifying sperm proteins and their corresponding genes. This was aided by the use of monoclonal antibodies that preferentially bind to yet unknown sperm molecules. By identifying their target molecules, this approach identified proteins with relevance to Mytilus sperm function. This procedure identified 16 proteins, for example, enkurin, laminin, porin and heat shock proteins. The potential use of these proteins as genetic markers to study reproductive isolation is exemplified by analysing the enkurin locus. Enkurin evolution is driven by purifying selection, the locus displays high levels of intraspecific variation and species-specific alleles group in distinct phylogenetic clusters. These findings characterize enkurin as informative candidate biomarker for analyses of clinal variation and differential introgression in hybrid zones, for example, to understand determinants of reproductive isolation in Baltic Mytilus populations.}, language = {en} }