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Hapten-specific single-cell selection of hybridoma clones by FACS for the generation of monoclonal antibodies Martin Dippong, Peter Carl, Christine Lenz, Jörg Schenk, Katrin Hoffmann, Timm Schwaar, Rudolf J. Schneider, and Maren Kuhne Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b04569 • Publication Date (Web): 10 Mar 2017 Downloaded from http://pubs.acs.org on March 11, 2017
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Hapten-specific single-cell selection of hybridoma clones by FACS for the generation of monoclonal antibodies. Martin Dippong†, ǂ, ⊥, Peter Carl†, ‡, Christine Lenz§, ∥, Jörg A. Schenk§, ∥, Katrin Hoffmann†, Timm Schwaar†, ‡, Rudolf J. Schneider†,* and Maren Kuhne†. † Bundesanstalt für Materialforschung und -prüfung (BAM), Richard-Willstätter-Str. 11, 12489 Berlin, Germany ǂ University of Potsdam, Institute for Biochemistry and Biology, Karl-Liebknecht-Str. 24-25, 14476 Potsdam, Germany ‡ Humboldt-Universität zu Berlin, Department of Chemistry, Brook-Taylor-Str. 2, 12489 Berlin § UP Transfer GmbH, Am Neuen Palais 10, 14469 Potsdam, Germany ∥ Hybrotec GmbH, Am Mühlenberg 11, 14476 Potsdam-Golm, Germany ⊥ Fraunhofer Institute for Cell Therapy and Immunology (IZI), Branch Bioanalytics and Bioprocesses Potsdam-Golm, Am Mühlenberg 13, 14476 Potsdam, Germany 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 eliminating the abovementioned 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-peroxidase conjugate (hapten-HRP), which is subsequently incubated with a fluorophorelabeled 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. 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 highthroughput technique for the generation of mAbs including increased yield of antibody-producing hybridoma clones, ensured monoclonality of sorted cells and reduced development times.
INTRODUCTION Hapten-specific monoclonal antibodies (mAbs) have become very important tools for immunoanalytical applications, especially for monitoring environmental contaminants like hormones,1 as well as mycotoxins in food2 and pesticides.3 For immunization, either high molecular weight compounds such as proteins, or low molecular weight compounds, haptens, can be addressed as targets. Proteins are used directly for immunization whereas haptens do not cause an immune response and have to be linked to a carrier protein like bovine serum albumin (BSA), ovalbumin (OVA) or keyhole limpet hemocyanin (KLH) to trigger an immune reaction.4 Then antibody-producing B cells are fused with myeloma cells in order to obtain hybridoma cells and are plated in multi-well plates.5 The subsequent identification and isolation of target-specific antibody-producing hybridoma clones is generally considered to be one of the most critical steps in antibody production. The percentage of analyte-specific hybridoma cells can be very low hence hundreds up to thousands of hybridoma cells have to be screened to identify a positive clone. The immunoassay-based screening is mainly done by (indirect) enzyme-linked immunosorbent assays (ELISA). The supernatant of each well is tested for target-specific antibodies. Cells of positive wells are subsequently diluted several times (limiting dilution) because desired target-specific hybridoma cells may be mixed
with other hybridoma cells in the same well and thus may be overgrown.6 Furthermore, an often occurring problem in haptenspecific antibody generation is the appearance of antibodies that are not directed against the hapten but against epitopes found on the carrier protein.7 This reduces the total amount of hybridoma cells that produce hapten-specific antibodies. Consequently, more cells have to be screened in order to obtain the desired hybridoma clone that produces a selective antibody against the hapten.8 For screening of supernatants, besides conventional ELISA, few sophisticated methods have been developed, among them Biacore Screening,9 phage display,10 antibody and antigen based microarrays11,12 as well as bio-layer interferometry13 and fluorescence polarisation immunoassay.14 In order to minimize or even avoid the effort of the limiting dilution with the subsequent screening of supernatants some methods have been developed to perform single-cell hybridoma screenings. Akselband et al. reported on encapsulation of single cells into agarose gel microdrops.15 Drawbacks of this method, however, are physical cell stress caused by a solid medium and low encapsulation efficiencies of cells (
