title: Cell surface-fluorescence immunosorbent assay for real-time detection of hybridomas with efficient antibody secretion at the single-cell level.
abstract: For establishing cells that secrete antibodies most efficiently (e.g., hybridomas, CHO (Chinese hamster ovary) cells), the screening and subsequent breeding of promising cells have been performed at the single-colony level, which requires several weeks to propagate a substantial number of cells by forming colonies from single cells for evaluation by the conventional assays. However, this screening process lacks high-throughput performance in time and colony numbers. Therefore, development of novel methods is expected to identify single cells secreting higher amounts of antibodies in real-time and in a nondestructive manner without colony formation. In this study, we prepared lipid-labeled antimouse IgG Fc antibodies (capture molecules) that were uniformly displayed on the surface of candidate cells. Secreted nascent antibodies were subsequently sandwiched between capture molecules and fluorescence-labeled antimouse IgG F(ab')(2) F(ab')(2) (detection molecules). This newly developed method is hereinafter referred to as a cell surface-fluorescence immunosorbent assay (CS-FIA). The fluorescence intensity of each cell was found to correlate well with the amount of sandwiched antibodies (from 6.25 fg/cell to 6.40 pg/cell). When about 4 × 10(3) cells of mouse hybridomas were subjected to CS-FIA, we isolated 28 hybridomas showing the highest fluorescence intensity within a day. Furthermore, after propagation of single cells to about 10(5) cells (after 2 weeks), 20 hybridomas were still able to secrete higher amounts (up to 7-fold) of antibodies than parental hybridomas. Our results demonstrate that CS-FIA is a powerful method for the single-cell-based establishment of cells that secrete most efficiently not only antibodies but also various biomolecules.
authors.affiliation: Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan.
authors.forename: Akiko
authors.initials: A
authors.lastname: Kida
authors.forename: Masumi
authors.initials: M
authors.lastname: Iijima
authors.forename: Tomoaki
authors.initials: T
authors.lastname: Niimi
authors.forename: Andrés D
authors.initials: AD
authors.lastname: Maturana
authors.forename: Nobuo
authors.initials: N
authors.lastname: Yoshimoto
authors.forename: Shun'ichi
authors.initials: S
authors.lastname: Kuroda
mesh_terms: D000818:Animals; D000906:Antibodies / Q000276:immunology / Q000378:metabolism*; D016466:CHO Cells; D002462:Cell Membrane / Q000276:immunology / Q000378:metabolism*; D003199:Computer Systems; D006224:Cricetinae; D003412:Cricetulus; D057809:HEK293 Cells; D006801:Humans; D006825:Hybridomas / Q000276:immunology / Q000378:metabolism*; D007163:Immunosorbent Techniques; D051379:Mice; D017452:Receptors, IgG / Q000276:immunology
chemical_list: D000906:Antibodies; C485701:Fcgr1 protein, mouse; D017452:Receptors, IgG