Fraunhofer Life Science Day

  • Their high affinity, selectivity and specificity make monoclonal antibodies (mAbs) very important tools in research, diagnostics and therapy. The production of mAbs is routinely performed by hybridoma technique. Hybridomas are generated by fusing antibody-producing B cells with indefinitely proliferating myeloma cells. Both, cell fusion and the indispensable identification and isolation of the desired antibody-producing hybridoma clone is still far from being trivial. We describe a novel method that should help to optimize the cell fusion, and the screening for antibody-secreting hapten-specific hybridoma cells by using fluorescence activated cell sorting and single-cell fusion. Hybridoma cells specific for a hapten were incubated with a hapten-peroxidase conjugate (hapten-HRP), which was subsequently incubated with a fluorophore-labeled polyclonal anti-peroxidase antibody (anti-HRP-Alexa Fluor® 488). To characterize the expression of membrane-bound immunoglobulin G (IgG) aTheir high affinity, selectivity and specificity make monoclonal antibodies (mAbs) very important tools in research, diagnostics and therapy. The production of mAbs is routinely performed by hybridoma technique. Hybridomas are generated by fusing antibody-producing B cells with indefinitely proliferating myeloma cells. Both, cell fusion and the indispensable identification and isolation of the desired antibody-producing hybridoma clone is still far from being trivial. We describe a novel method that should help to optimize the cell fusion, and the screening for antibody-secreting hapten-specific hybridoma cells by using fluorescence activated cell sorting and single-cell fusion. Hybridoma cells specific for a hapten were incubated with a hapten-peroxidase conjugate (hapten-HRP), which was subsequently incubated with a fluorophore-labeled polyclonal anti-peroxidase antibody (anti-HRP-Alexa Fluor® 488). To characterize the expression of membrane-bound immunoglobulin G (IgG) a fluorophore-labeled anti-mouse IgG antibody (anti-IgG-Alexa Fluor® 647) was used. Successful staining was verified by confocal laser scanning microscopy (CLSM). We show that it is possible to specifically label hapten-specific hybridoma cells. It should also be possible to use this labelling approach for the isolation of hapten-specific B cells from the spleen and use these cells for single-cell fusion. Parallelized cell fusion chips were developed to increase fusion efficiencies.zeige mehrzeige weniger

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Metadaten
Autoren/innen:Martin Dippong
Koautoren/innen:C. Gürnth-Marschner, M. Kirschbaum, C. Duschl, Rudolf Schneider, Maren Kuhne, Katrin Hoffmann
Dokumenttyp:Posterpräsentation
Veröffentlichungsform:Präsentation
Sprache:Englisch
Jahr der Erstveröffentlichung:2015
Organisationseinheit der BAM:1 Analytische Chemie; Referenzmaterialien
1 Analytische Chemie; Referenzmaterialien / 1.0 Abteilungsleitung und andere
DDC-Klassifikation:Naturwissenschaften und Mathematik / Chemie / Analytische Chemie
Freie Schlagwörter:Hybridoma; Monoclonal antibodies; Single-cell fusion
Themenfelder/Aktivitätsfelder der BAM:Analytical Sciences
Veranstaltung:Fraunhofer Life Science Day 2015
Veranstaltungsort:Leipzig, Germany
Beginndatum der Veranstaltung:03.07.2015
Verfügbarkeit des Dokuments:Datei im Netzwerk der BAM verfügbar ("Closed Access")
Datum der Freischaltung:19.10.2016
Referierte Publikation:Nein