Reference - Detail
| RRC ID | 44231 |
|---|---|
| Author | Kida A, Iijima M, Niimi T, Maturana AD, Yoshimoto N, Kuroda S. |
| Title | Cell surface-fluorescence immunosorbent assay for real-time detection of hybridomas with efficient antibody secretion at the single-cell level. |
| Journal | Anal Chem |
| 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. |
| Volume | 85(3) |
| Pages | 1753-9 |
| Published | 2013-2-5 |
| DOI | 10.1021/ac303067k |
| PMID | 23297690 |
| MeSH | Animals Antibodies / immunology Antibodies / metabolism* CHO Cells Cell Membrane / immunology Cell Membrane / metabolism* Computer Systems* Cricetinae Cricetulus HEK293 Cells Humans Hybridomas / immunology Hybridomas / metabolism* Immunosorbent Techniques Mice Receptors, IgG / immunology |
| IF | 6.785 |
| Times Cited | 12 |
| WOS Category | CHEMISTRY, ANALYTICAL |
| Altmetric score |
オルトメトリクス指標項目
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| The most frequently cited source | Patent(IFI CLAIMS) |
| Total number of mentions | 2 |
| Altmetric score changes over past 6months | 0.0 |
| Resource | |
| Human and Animal Cells | HyLDH·YK-1(RCB0709) |