Quick enriching and separating method of natural killer cells in peripheral blood of human

Abstract

The invention discloses a method for separating and enriching natural killer cells (natural killer cells, NK) in peripheral blood for further providing a basis for subsequent study of natural killer cells, and relates to the biomedical field. The method comprises the following steps of: covalent coupling of dendritic polymers and mouse anti-human NKp46 antibodies; further coating long-chain biotin molecules by dendritic polymers modified by mouse anti-human NKp46 antibodies; capturing NK in a peripheral blood sample by dendritic polymers co-modified by mouse anti-human NKp46 antibodies and long-chain biotin; identifying and coupling long-chain dendritic polymers in the peripheral blood by streptavidin modified nanomagnetic beads; and separating and resuspending the captured NK. The resuspension can be directly used for subsequent analysis. Compared with conventional cell separation methods, the method is more suitable for magnetic separation of NK in complex peripheral blood samples, so that the magnetic separation time is shortened, and the NK separation efficiency in the peripheral blood sample is improved.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a method for separating natural killer cells based on nano magnetic beads. Background technique [0002] Natural killer cells (NK) are important immune cells in the body, not only related to anti-tumor, anti-viral infection and immune regulation, but also involved in the occurrence of hypersensitivity and autoimmune diseases in some cases. NK cells are the first line of defense of the body's non-specific immunity, but due to the separation and purification of NK cells and the conditions of in vitro culture, the research on them is limited. At present, a variety of methods for NK cell purification have been established at home and abroad, but their high cost, complicated operation and other factors limit their popularization and application in China. However, the number of NK cells in peripheral blood or splenic lymphocytes is very small, and it is difficult to obtain a large number of...

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 cells in the food matrix; 3) The micron magnetic beads have poor monodispersity and are prone to self-aggregation or self-aggregation in the peripheral 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 blood cell concentration of non-natural killer cells is large, micron magnetic beads are prone to non-specific adsorption, and it is difficult to achieve specificity for natural killer cells in blood Separation; 6) Excessive concentration of micron magnetic beads will cause damage to natural killer cells (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

Images