40320 2017 eng 4007 4012 7 89 article ACS Publications 1 -- -- -- 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. Analytical Chemistry 10.1021/acs.analchem.6b04569 0003-2700 1520-6882 23.05.2017 Martin Dippong Peter Carl C. Lenz J. A. Schenk Katrin Hoffmann Timm Schwaar Rudolf Schneider Maren Kuhne eng uncontrolled Monoclonal antibodies eng uncontrolled FACS eng uncontrolled Hybridoma cells Analytische Chemie Sanitär- und Kommunaltechnik; Umwelttechnik Verlagsliteratur Datei im Netzwerk der BAM verfügbar ("Closed Access")