TY - JOUR A1 - Schlör, Anja A1 - Holzlöhner, Pamela A1 - Listek, Martin A1 - Grieß, Cindy A1 - Butze, Monique A1 - Micheel, Burkhard A1 - Hentschel, Christian A1 - Sowa, Mandy A1 - Roggenbuck, Dirk A1 - Schierack, Peter A1 - Füner, Jonas A1 - Schliebs, Erik A1 - Goihl, Alexander A1 - Reinhold, Dirk A1 - Hanack, Katja T1 - Generation and validation of murine monoclonal and camelid recombinant single domain antibodies specific for human pancreatic glycoprotein 2 JF - New biotechnology N2 - 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. KW - glycoprotein GP2 KW - Monoclonal antibodies KW - Camelid single domain antibodies Y1 - 2018 U6 - https://doi.org/10.1016/j.nbt.2018.03.006 SN - 1871-6784 SN - 1876-4347 VL - 45 SP - 60 EP - 68 PB - Elsevier CY - Amsterdam ER - TY - JOUR A1 - Holzlöhner, Pamela A1 - Butze, Monique A1 - Maier, Natalia A1 - Hebel, Nicole A1 - Schliebs, Erik A1 - Micheel, Burkhard A1 - Fuener, Jonas A1 - Heidicke, Gabriele A1 - Hanack, Katja T1 - Generation of murine monoclonal antibodies with specificity against conventional camelid IgG1 and heavy-chain only IgG2/3 JF - Veterinary Immunology and Immunopathology N2 - 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. KW - Camelid antibodies KW - Heavy-chain only antibodies KW - Monoclonal antibodies KW - Secondary antibodies KW - Vaccination KW - Disease monitoring Y1 - 2018 U6 - https://doi.org/10.1016/j.vetimm.2018.01.006 SN - 0165-2427 SN - 1873-2534 VL - 197 SP - 1 EP - 6 PB - Elsevier CY - Amsterdam ER - TY - JOUR A1 - Holzlöhner, Pamela A1 - Hanack, Katja T1 - Generation of murine monoclonal antibodies by hybridoma technology JF - JoVE : Video journal N2 - 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. KW - Immunology KW - Issue 119 KW - monoclonal antibodies KW - hybridoma technology KW - myeloma cells KW - B lymphocytes KW - antigen KW - immunconjugate Y1 - 2017 U6 - https://doi.org/10.3791/54832 SN - 1940-087X IS - 119 PB - JoVE CY - Cambridge ER - TY - GEN A1 - Maier, Natalia A1 - Holzlöhner, Pamela A1 - Hoenow, Anja A1 - Scheunemann, Astrid A1 - Weschke, Daniel A1 - Hanack, Katja T1 - Characterization of monoclonal antibodies generated by in vitro immunization T2 - The journal of immunology N2 - 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. Y1 - 2016 SN - 0022-1767 SN - 1550-6606 VL - 196 PB - American Assoc. of Immunologists CY - Bethesda ER - TY - GEN A1 - Holzlöhner, Pamela A1 - Butze, Monique A1 - Hebel, Nicole A1 - Weschke, Daniel A1 - Schliebs, E. A1 - Naumann, F. A1 - Füner, J. A1 - Micheel, Burkhard A1 - Hanack, Katja T1 - Monoclonal mouse antibodies against PBMC subpopulations of New World camelides T2 - European journal of immunology Y1 - 2016 SN - 0014-2980 SN - 1521-4141 VL - 46 SP - 1175 EP - 1175 PB - Wiley-Blackwell CY - Hoboken ER - TY - CHAP A1 - Holzlöhner, Pamela A1 - Schliebs, Erik A1 - Maier, Natalia A1 - Füner, Jonas A1 - Micheel, Burkhard A1 - Heilmann, Katja T1 - Production of monoclonal camelid antibodies by means of hybridoma technology T2 - The journal of immunology Y1 - 2013 SN - 0022-1767 VL - 190 PB - American Assoc. of Immunologists CY - Bethesda ER - TY - CHAP A1 - Heilmann, Katja A1 - Wand, Inga A1 - Holzlöhner, Pamela A1 - Micheel, Burkhard T1 - Cooperation of dendritic cells with naive lymphocyte populations to induce the generation of antigen-specific antibodies in vitro T2 - The journal of immunology Y1 - 2012 SN - 0022-1767 VL - 188 IS - 6 PB - American Assoc. of Immunologists CY - Bethesda ER - TY - JOUR A1 - Wand, Inga A1 - Holzlöhner, Pamela A1 - Neupert, Steffi A1 - Micheel, Burkhard A1 - Heilmann, Katja T1 - Cooperation of dendritic cells with naive lymphocyte populations to induce the generation of antigen-specific antibodies in vitro JF - Journal of biotechnology N2 - 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. KW - In vitro immunization KW - Activation of dendritic cells KW - Interaction of T and B cells with antigen-presenting cells KW - Induction of antibody responses Y1 - 2011 U6 - https://doi.org/10.1016/j.jbiotec.2011.09.002 SN - 0168-1656 VL - 156 IS - 3 SP - 173 EP - 181 PB - Elsevier CY - Amsterdam ER -