Magnetic Cell Separation Using Antibody Binding with Protein A Expressed on Bacterial Magnetic Particles

Abstract

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 approximately 97.5 +/- 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 approximately 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 TNFalpha and IL6 were slightly induced in the absence of LPS in the positive fraction.

Formation of magnetite by bacteria and its application

“…The procedure for positive selection involves incubation of mononuclear cells and mouse monoclonal antibodies against different cell surface antigens (CD8, CD14, CD19 and CD20) prior to treatment with protein A-BacMP complexes with rabbit anti-mouse IgG secondary antibodies. The average purities of the separated mononuclear cells of CD19 C and CD20 C were 97.5 and 97.6%, respectively (Kuhara et al 2004). Furthermore, more than 95% of the recovery ratio was obtained for these cells.…”

“…Because BacMPs consist of ferromagnetic iron oxide, they are easily separated from cell suspensions using a permanent magnet with no special column. We have therefore applied BacMPs to develop highly efficient magnetic cell separation (Kuhara et al 2004). BacMPs expressing protein A (protein A-BacMPs) bound to the Fc fragment of anti-mouse IgG antibodies were used to separate mononuclear cells from peripheral blood.…”

The Potential of Genetically Engineered Magnetic Particles in Biomedical Applications

“…To enrich for target cells, cell surface antigens, such as cluster of differentiation (CD) antigens, were used as markers. CD8, CD14, CD19, CD20, and CD34 positive cells were efficiently enriched from peripheral blood (Kuhara et al, 2004;Matsunaga et al, 2006). The separated CD34 positive cells retained the capability of forming colonies as hematopoietic stem cells.…”

“…The major advantage of magnetic particles is that they can be easily manipulated by magnetic force, which enables rapid and easy separation of target molecules bound to the particles from reaction mixtures (Mirzabekov et al, 2000;Gu et al, 2003;Kuhara et al, 2004;Xu et al, 2004). Use of magnetic particles is beneficial for complete automation of steps, resulting in minimal manual labor and providing more precise results (Sawakami-Kobayashi et al, 2003).…”

Separation of Tricomponent Protein Mixtures with Triblock Nanorods

“…Protein release is effected by a change in pH or by eluting the column with a solution with a high concentration of imidazole. [12][13][14] Nanomaterials such as nanoparticles, nanotubes, and nanorods have been extensively used as complementary candidates for the effective separation of biomaterials [25][26][27][28][29][30] as well as in the field of biomedical diagnostics and therapeutics. [31][32][33][34][35][36][37] Previously we reported that twocomponent triblock magnetic nanorods with Au end blocks and a Ni interior block can be used as affinity templates for simple and efficient separation of His-tagged proteins from other proteins without affinity tags.…”