Quick separation method of dendritic cells

Abstract

The invention discloses a method for separating and enriching dendritic cells (Dendritice cells, DC) in whole blood for further providing a basis for subsequent study of dendritic cells, and relates to the biomedical field. The method comprises the following steps of: covalent coupling of hyperbranched polymers and mouse anti-human dendritic cell monoclonal antibodies; further coating long-chain biotin molecules by hyperbranched polymers modified by mouse anti-human dendritic cell monoclonal antibodies; capturing DC in a whole blood sample by hyperbranched polymers co-modified by mouse anti-human dendritic cell monoclonal antibodies and long-chain biotin; identifying and coupling long-chain biotin hyperbranched polymers in the whole blood by streptavidin modified nanomagnetic beads; and separating and resuspending the captured DC. The resuspension can be directly used for subsequent analysis. Compared with conventional cell separation methods, the method is more suitable for magnetic separation of DC in complex whole blood samples, so that the magnetic separation time is shortened, and the DC separation efficiency in the whole blood sample is improved.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a method for separating dendritic cells based on nano magnetic beads. Background technique [0002] Dendritic cells (dendritic cells, DCs) were discovered by American scholar Steinman in 1973. They are the most powerful and only professional antigen presenting cells (antigen presenting cells, APCs) that can activate naive T cells. , its quantity and mature state can determine the immune status of the body, so its in-depth study has important theoretical and practical significance. DC is operable in vitro, so obtaining a certain amount of functional DC is the key to in-depth study of its biological characteristics. However, the content of DC in tissues is very small, accounting for only 0.16-0.68% of peripheral blood leukocytes and 0.5-1.6% of monocytes in blood. The existing methods cannot directly detect trace amounts of DC in peripheral blood. Therefore, the high-efficiency enrichm...

AI Technical Summary

Problems solved by technology

However, the current separation technology based on micron-scale immunomagnetic beads has many limitations: 1) The specific surface area of ​​micron-sized magnetic beads is relatively small, which reduces the capture efficiency of magnetic beads; The combination of cells through a multiphase reaction usually takes longer to specifically capture the cells in the food matrix; 3) The micron magnetic beads have poor monodispersity and are prone to self-aggregation or self-aggregation in the whole blood matrix Precipitation; 4) The traditional immunomagnetic separation technology often directly couples the antibody to the immunomagnetic beads. This process often leads to a greatly reduced activity of the antibody and a change in the spatial direction of the antibody, which increases the inter-antibody interaction. Steric hindrance effect, which reduces the capture efficiency of antibodies 5) Blood viscosity is high and the concentration of non-DC blood cells is large, micron magnetic beads are prone to non-specific adsorption, and it is difficult to achieve specific separation of DC in blood; 6) Excessive concentration of micron magnetic beads will cause DC damage (the magnetic field causes the magnetic beads on the cell surface to attract each other, causing the cells to be squeezed or even ruptured), resulting in failure of separation

The distance between the antibody and the surface of the magnetic bead is too close, the nature of the magnetic bead itself and the residual hydrophobic or strong hydrophilic groups on the surface are likely to cause changes in the spatial conformation of the antibody, resulting in a decrease in the biological activity of the antibody

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