@inproceedings{HolzloehnerSchliebsMaieretal.2013, author = {Holzl{\"o}hner, Pamela and Schliebs, Erik and Maier, Natalia and F{\"u}ner, Jonas and Micheel, Burkhard and Heilmann, Katja}, title = {Production of monoclonal camelid antibodies by means of hybridoma technology}, series = {The journal of immunology}, volume = {190}, booktitle = {The journal of immunology}, publisher = {American Assoc. of Immunologists}, address = {Bethesda}, issn = {0022-1767}, pages = {1}, year = {2013}, language = {en} } @inproceedings{HeilmannWandHolzloehneretal.2012, author = {Heilmann, Katja and Wand, Inga and Holzl{\"o}hner, Pamela and Micheel, Burkhard}, title = {Cooperation of dendritic cells with naive lymphocyte populations to induce the generation of antigen-specific antibodies in vitro}, series = {The journal of immunology}, volume = {188}, booktitle = {The journal of immunology}, number = {6}, publisher = {American Assoc. of Immunologists}, address = {Bethesda}, issn = {0022-1767}, pages = {1}, year = {2012}, 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{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} } @misc{MaierHolzloehnerHoenowetal.2016, author = {Maier, Natalia and Holzl{\"o}hner, Pamela and Hoenow, Anja and Scheunemann, Astrid and Weschke, Daniel and Hanack, Katja}, title = {Characterization of monoclonal antibodies generated by in vitro immunization}, series = {The journal of immunology}, volume = {196}, journal = {The journal of immunology}, publisher = {American Assoc. of Immunologists}, address = {Bethesda}, issn = {0022-1767}, pages = {2}, year = {2016}, abstract = {Monoclonal antibodies are highly valuable tools in biomedicine but the generation by hybridoma technology is very time-consuming and elaborate. In order to circumvent the consisting drawbacks an in vitro immunization approach was established by which murine as well as human monoclonal antibodies against a viral coat protein could be developed. The in vitro immunization process was performed by isolation of murine hematopoietic stem cells or human monocytes and an in vitro differentiation into immature dendritic cells. After antigen loading the cells were co-cultivated with naive T and B lymphocytes for three days in order to obtain antigen-specific B lymphocytes in culture, followed by fusion with murine myeloma cells or human/murine heteromyeloma cells. Antigen-specific hybridomas were selected and the generated antibodies were purified and characterized in this study by ELISA, western blot, gene sequencing, affinity measurements. Further the characteristics were compared to a monoclonal antibody against the same target generated by conventional hybridoma technology. Isotype detection revealed a murine IgM and a human IgG4 antibody in comparison to an IgG1 for the conventionally generated antibody. The antibodies derived from in vitro immunization showed indeed a lower affinity for the antigen as compared to the conventionally generated one, which is probably based on the significantly shorter B cell maturation (3 days) during the immunization process. Nevertheless, they were suitable for building up a sandwich based detection system. Therefore, the in vitro immunization approach seems to be a good and particularly fast alternative to conventional hybridoma technology.}, 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} } @misc{HolzloehnerButzeHebeletal.2016, author = {Holzl{\"o}hner, Pamela and Butze, Monique and Hebel, Nicole and Weschke, Daniel and Schliebs, E. and Naumann, F. and F{\"u}ner, J. and Micheel, Burkhard and Hanack, Katja}, title = {Monoclonal mouse antibodies against PBMC subpopulations of New World camelides}, series = {European journal of immunology}, volume = {46}, journal = {European journal of immunology}, publisher = {Wiley-Blackwell}, address = {Hoboken}, issn = {0014-2980}, pages = {1175 -- 1175}, year = {2016}, language = {en} } @article{HolzloehnerHanack2017, author = {Holzl{\"o}hner, Pamela and Hanack, Katja}, title = {Generation of murine monoclonal antibodies by hybridoma technology}, series = {JoVE : Video journal}, journal = {JoVE : Video journal}, number = {119}, publisher = {JoVE}, address = {Cambridge}, issn = {1940-087X}, doi = {10.3791/54832}, pages = {7}, year = {2017}, abstract = {Monoclonal antibodies are universal binding molecules and are widely used in biomedicine and research. Nevertheless, the generation of these binding molecules is time-consuming and laborious due to the complicated handling and lack of alternatives. The aim of this protocol is to provide one standard method for the generation of monoclonal antibodies using hybridoma technology. This technology combines two steps. Step 1 is an appropriate immunization of the animal and step 2 is the fusion of B lymphocytes with immortal myeloma cells in order to generate hybrids possessing both parental functions, such as the production of antibody molecules and immortality. The generated hybridoma cells were then recloned and diluted to obtain stable monoclonal cell cultures secreting the desired monoclonal antibody in the culture supernatant. The supernatants were tested in enzyme-linked immunosorbent assays (ELISA) for antigen specificity. After the selection of appropriate cell clones, the cells were transferred to mass cultivation in order to produce the desired antibody molecule in large amounts. The purification of the antibodies is routinely performed by affinity chromatography. After purification, the antibody molecule can be characterized and validated for the final test application. The whole process takes 8 to 12 months of development, and there is a high risk that the antibody will not work in the desired test system.}, language = {en} }