TY - JOUR A1 - Dippong, Martin A1 - Carl, Peter A1 - Lenz, C. A1 - Schenk, J. A. A1 - Hoffmann, Katrin A1 - Schwaar, Timm A1 - Schneider, Rudolf A1 - Kuhne, Maren T1 - Hapten-specific single-cell selection of hybridoma clones by fluorescence-activated cell sorting for the generation of monoclonal antibodies T2 - Analytical Chemistry N2 - 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. PB - ACS Publications KW - Monoclonal antibodies KW - FACS KW - Hybridoma cells PY - 2017 UR - https://opus4.kobv.de/opus4-bam/frontdoor/index/index/docId/40320 AN - OPUS4-40320 SN - 0003-2700 SN - 1520-6882 VL - 89 IS - 7 SP - 4007 EP - 4012 AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany