Magnetic cell separation using antibody binding with protein a expressed on bacterial magnetic particles
Magnetic cell separation using antibody binding with protein a expressed on bacterial magnetic particles
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DOI：
10.1021/ac0493727
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发表时间：
2004-11-01
影响因子：
7.4
通讯作者：
Matsunaga, T
中科院分区：
文献类型：
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作者：
Kuhara, M;Takeyama, H;Matsunaga, T
细菌磁性颗粒 (BacMP) 是用于表面显示系统的高效蛋白质平台。在本研究中，使用在 BacMP 膜表面表达蛋白 A 的 BacMP（蛋白 A-BacMP）分离外周血中的单核细胞，该蛋白与抗小鼠 IgG 抗体的 Fc 片段复合。阳性选择的过程包括孵育单核细胞和针对不同细胞表面抗原（CD8、CD14、CD19、CD20）的小鼠单克隆抗体，然后用与兔抗小鼠 IgG 二抗结合的蛋白 A-BacMP 进行处理。流式细胞术分析显示磁分离后阳性部分中有97.5±1.7%的CD19(+)和CD20(+)细胞。阴性部分中阴性细胞的比例为-97.6+/-1.4%。这表明可以有效地将CD19(+)和CD20(+)细胞与单核细胞分离。还使用蛋白 A-BacMP 与抗体结合来分离干细胞标记 (CD34) 阳性细胞。 May-Grunwald Giemsa 染色显示出较高的核/细胞质比，这表明干细胞的典型染色模式。分离的细胞具有作为造血干细胞的集落形成的能力。此外，当细胞在有或没有脂多糖(LPS)的情况下培养时，通过ELISA测量培养上清液中的细胞因子来评价磁性细胞分离对CD14(+)细胞的抑制效果。在所有级分中存在 1 ng/mL LPS 时，观察到 IL1-β、TNFα 和 IL6 的诱导。另一方面，在不存在LPS的情况下，BacMPs对CD14(+)细胞以及人工磁性颗粒几乎没有免疫增强作用，尽管在阳性部分中不存在LPS的情况下TNFa和IL6被轻微诱导。
Bacterial magnetic particles (BacMPs) are efficient platforms of proteins for surface display systems. In this study, mononuclear cells from peripheral blood were separated using BacMPs expressing protein A on the BacMP membrane surface (protein A-BacMPs), which were complexed with the Fc fragment of anti-mouse IgG antibody. The procedure of positive selection involves incubation of mononuclear cells and mouse monoclonal antibodies against different cell surface antigens (CD8, CD14, CD19, CD20) prior to treatment with protein A-BacMP binding with rabbit anti-mouse IgG secondary antibodies. Flow cytometric analysis showed that similar to97.5 +/- 1.7% of CD19(+) and CD20(+) cells were involved in the positive fraction after magnetic separation. The ratio of the negative cells in the negative fraction was -97.6 +/- 1.4%. This indicates that CD19(+) and CD20(+) cells can be efficiently separated from mononuclear cells. Stem cell marker (CD34) positive cells were also separated using protein A-BacMP binding with antibody. May-Grunwald Giemsa stain showed a high nuclear/cytoplasm ratio, which indicates a typical staining pattern of stem cells. The separated cells had the capability of colony formation as hematopoietic stem cells. Furthermore, the inhibitory effect of magnetic cell separation on CD14(+) cells was evaluated by measurement of cytokine in the culture supernatant by ELISA when the cells were cultured with or without lipopolysaccharide (LPS). The induction of IL1-beta, TNFalpha, and IL6 was observed in the presence of 1 ng/mL LPS in all fractions. On the other hand, in the absence of LPS, BacMPs had little immunopotentiation to CD14(+) cells as well as that of artificial magnetic particles, although TNFa and IL6 were slightly induced in the absence of LPS in the positive fraction.