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Hapten-Specific Single-Cell Selection of Hybridoma Clones by Fluorescence-Activated Cell Sorting for the Generation of Monoclonal Antibodies

: Dippong, M.; Carl, P.; Lenz, C.; Schenk, J.A.; Hoffmann, K.; Schwaar, T.; Schneider, R.J.; Kuhne, M.


Analytical chemistry 89 (2017), No.7, pp.4007-4012
ISSN: 0003-2700
ISSN: 1520-6882
Journal Article
Fraunhofer IZI ()

The conventional hybridoma screening and subdoning 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 cell's 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-hapteri 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.