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Autor

  • Carl, Peter (1)
  • Dippong, Martin (1)
  • Hoffmann, Katrin (1)
  • Kuhne, Maren (1)
  • Lenz, C. (1)
  • Schenk, J. A. (1)
  • Schneider, Rudolf J. (1)
  • Schwaar, Timm (1)

Erscheinungsjahr

  • 2017 (1) (entfernen)

Schlagworte

  • FACS (1)
  • Hybridoma cells (1)
  • Monoclonal antibodies (1)

Organisationseinheit der BAM

  • 1.2 Biophotonik (1) (entfernen)

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Hapten-specific single-cell selection of hybridoma clones by fluorescence-activated cell sorting for the generation of monoclonal antibodies (2017)
Dippong, Martin ; Carl, Peter ; Lenz, C. ; Schenk, J. A. ; Hoffmann, Katrin ; Schwaar, Timm ; Schneider, Rudolf J. ; Kuhne, Maren
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.
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