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

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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 ∼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 ∼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-β, TNFα, 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 TNFα and IL6 were slightly induced in the absence of LPS in the positive fraction.