Cell Surface-Fluorescence Immunosorbent Assay for Real-Time Detection of Hybridomas with Efficient Antibody Secretion at the Single-Cell Level
Cell Surface-Fluorescence Immunosorbent Assay for Real-Time Detection of Hybridomas with Efficient Antibody Secretion at the Single-Cell Level
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DOI：
10.1021/ac303067k
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发表时间：
2013-02-05
影响因子：
7.4
通讯作者：
Kuroda, Shun'ichi
中科院分区：
文献类型：
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作者：
Kida, Akiko;Iijima, Masumi;Kuroda, Shun'ichi
为了建立最有效分泌抗体的细胞（例如，杂交瘤，CHO（中国仓鼠卵巢）细胞），筛选和随后的有希望的细胞育种已经在单菌落水平上进行，这需要几周的时间来繁殖大量的细胞，从单个细胞形成菌落，以供常规试验评估。然而，这种筛选方法在时间和菌落数量上缺乏高通量性能。因此，新方法的发展有望识别单细胞分泌更多的抗体实时和非破坏性的方式，没有集落形成。在这项研究中，我们制备了脂质标记的抗小鼠IgG Fc抗体（捕获分子），该抗体均匀地显示在候选细胞表面。随后，将分泌的新生抗体夹在捕获分子和荧光标记的抗小鼠IgG F(ab')(2)F(ab')(2)（检测分子）之间。这种新开发的方法以下称为细胞表面荧光免疫吸附试验（CS-FIA）。发现每个细胞的荧光强度与夹心抗体的数量有很好的相关性（从6.25 fg/cell到6.40 pg/cell）。CS-FIA处理小鼠杂交瘤细胞约4 × 10(3)个，在一天内分离出28个荧光强度最高的杂交瘤细胞。此外，在单细胞繁殖到约10(5)个细胞后（2周后），20个杂交瘤仍然能够分泌比亲本杂交瘤更高数量的抗体（高达7倍）。我们的研究结果表明，CS-FIA是一种基于单细胞的细胞构建的强大方法，不仅可以最有效地分泌抗体，还可以分泌各种生物分子。
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 X 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.