Cell surface-fluorescence immunosorbent assay for real-time detection of hybridomas with efficient antibody secretion at the single-cell level

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.