title: Hapten-Specific Single-Cell Selection of Hybridoma Clones by Fluorescence-Activated Cell Sorting for the Generation of Monoclonal Antibodies.
abstract: The conventional hybridoma screening and subcloning process is generally considered to be one of the most critical steps in hapten-specific antibody production. It is time-consuming, monoclonality is not guaranteed, and the number of clones that can be screened is limited. Our approach employs a novel hapten-specific labeling technique of hybridoma cells. This allows for fluorescence-activated cell sorting (FACS) and single-cell deposition and thereby eliminates the above-mentioned problems. A two-step staining approach is used to detect antigen specificity and antibody expression: in order to detect antigen specificity, hybridoma cells are incubated with a hapten-horseradish peroxidase conjugate (hapten-HRP), which is 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) is used. Hundreds of labeled hybridoma cells producing monoclonal antibodies (mAbs) specific for a hapten were rapidly isolated and deposited from a fusion mixture as single-cell clones via FACS. Enzyme-linked immunosorbent assay (ELISA) measurements of the supernatants of the sorted hybridoma clones revealed that all hapten-specific hybridoma clones secrete antibodies against the target. There are significant improvements using this high-throughput technique for the generation of mAbs including increased yield of antibody-producing hybridoma clones, ensured monoclonality of sorted cells, and reduced development times.
authors.affiliation: Bundesanstalt für Materialforschung und -prüfung (BAM) , Richard-Willstätter-Str. 11, 12489 Berlin, Germany.|University of Potsdam , Institute for Biochemistry and Biology, Karl-Liebknecht-Str. 24-25, 14476 Potsdam, Germany.|Fraunhofer Institute for Cell Therapy and Immunology (IZI) , Branch Bioanalytics and Bioprocesses Potsdam-Golm, Am Mühlenberg 13, 14476 Potsdam, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Dippong
authors.affiliation: Bundesanstalt für Materialforschung und -prüfung (BAM) , Richard-Willstätter-Str. 11, 12489 Berlin, Germany.|Humboldt-Universität zu Berlin , Department of Chemistry, Brook-Taylor-Str. 2, 12489 Berlin, Germany.
authors.forename: Peter
authors.initials: P
authors.lastname: Carl
authors.affiliation: UP Transfer GmbH , Am Neuen Palais 10, 14469 Potsdam, Germany.|Hybrotec GmbH , Am Mühlenberg 11, 14476 Potsdam-Golm, Germany.
authors.forename: Christine
authors.initials: C
authors.lastname: Lenz
authors.affiliation: UP Transfer GmbH , Am Neuen Palais 10, 14469 Potsdam, Germany.|Hybrotec GmbH , Am Mühlenberg 11, 14476 Potsdam-Golm, Germany.
authors.forename: Jörg A
authors.initials: JA
authors.lastname: Schenk
authors.affiliation: Bundesanstalt für Materialforschung und -prüfung (BAM) , Richard-Willstätter-Str. 11, 12489 Berlin, Germany.
authors.forename: Katrin
authors.initials: K
authors.lastname: Hoffmann
authors.affiliation: Bundesanstalt für Materialforschung und -prüfung (BAM) , Richard-Willstätter-Str. 11, 12489 Berlin, Germany.|Humboldt-Universität zu Berlin , Department of Chemistry, Brook-Taylor-Str. 2, 12489 Berlin, Germany.
authors.forename: Timm
authors.initials: T
authors.lastname: Schwaar
authors.affiliation: Bundesanstalt für Materialforschung und -prüfung (BAM) , Richard-Willstätter-Str. 11, 12489 Berlin, Germany.
authors.forename: Rudolf J
authors.identifier: 0000-0003-2228-1248
authors.initials: RJ
authors.lastname: Schneider
authors.affiliation: Bundesanstalt für Materialforschung und -prüfung (BAM) , Richard-Willstätter-Str. 11, 12489 Berlin, Germany.
authors.forename: Maren
authors.initials: M
authors.lastname: Kuhne
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000276:immunology*; D002469:Cell Separation; D002999:Clone Cells / Q000166:cytology / Q000276:immunology; D005434:Flow Cytometry; D006241:Haptens / Q000737:chemistry*; D006825:Hybridomas / Q000166:cytology* / Q000276:immunology; D051379:Mice; D059010:Single-Cell Analysis
chemical_list: D000911:Antibodies, Monoclonal; D006241:Haptens