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 - 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 -