Effects of antibody concentration on the separation of human natural killer cells in a commercial immunomagnetic separation system
Effects of antibody concentration on the separation of human natural killer cells in a commercial immunomagnetic separation system
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DOI：
10.1002/1097-0320(20011201)45:4
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发表时间：
2001-12-01
期刊：
影响因子：
--
通讯作者：
Chalmers, JJ
中科院分区：
文献类型：
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作者：
Comella, K;Nakamura, M;Chalmers, JJ
背景：基于细胞表面标记的细胞群的磁分离是许多生物和临床实验室的关键步骤。在本研究中，研究了抗体浓度对商业免疫磁性细胞分离系统中人自然杀伤细胞分离的影响。方法：具体而言，使用两步抗体夹心法对抗体结合位点的饱和度进行定量。第一步，即抗 CD56-PE 一抗的定量是通过使用流式细胞仪测量荧光强度来实现的。第二步，即抗 PE 微珠抗体试剂，是通过使用细胞追踪测速仪进行磁泳迁移率测量来实现的。 结果：根据这些研究的结果，使用两种不同的标记方案通过 Miltenyi Biotech Mini-MACS 细胞分离系统从人类外周血中分离 CD56+ 细胞。这两个标签协议中的第一个基于公司的建议，而第二个基于饱和研究的结果。这些研究的结果表明，磁泳迁移率是一抗和二抗浓度的函数，并且迁移率确实对分离系统的性能有影响。结论：随着迁移率因结合抗体的增加而增加，阳性细胞几乎完全从阴性洗脱液中消除。然而，随着结合抗体的增加以及因此流动性的增加，回收的阳性细胞的总量减少，推测这些细胞不可逆地保留在柱中。这些结果证明了免疫磁性细胞分离的复杂性以及进一步优化细胞分离过程的需要。 (C) 2001 Wiley-Liss, Inc.
Background: The magnetic separation of a cell population based on cell surface markers is a critical step in many biological and clinical laboratories. In this study, the effect of antibody concentration on the separation of human natural killer cells in a commercial, immunomagnetic cell separation system was investigated.Methods: Specifically, the degree of saturation of antibody binding sites using a two-step antibody sandwich was quantified. The quantification of the first step, a primary anti-CD56-PE antibody, was achieved through fluorescence intensity measurements using a flow cytometer. The quantification of the second step, an anti-PE-microbeads antibody reagent, was achieved through magnetophoretic mobility measurements using cell tracking velocimetry.Results: From the results of these studies, two different labeling protocols were used to separate CD56+ cells from human, peripheral blood by a Miltenyi Biotech Mini-MACS cell separation system. The first of these two labeling protocols was based on company recommendations, whereas the second was based on the results of the saturation studies. The results from these studies demonstrate that the magnetophoretic mobility is a function of both primary and secondary antibody concentrations and that mobility does have an effect on the performance of the separation system.Conclusions: As the mobility, increased due to an increase in bound antibodies, the positive cells were almost completely eliminated from the negative eluent. However, with an increase in bound antibodies, and thus mobility, the total amount of positive cells recovered decreases, It is speculated that these cells are irreversibly retained in the column. These results demonstrate the complexity of immunomagnetic cell separation and the need to further optimize the cell separation process. (C) 2001 Wiley-Liss, Inc.