Immunomagnetic microspheres are mainly used for cell separation. Immunomagnetic microspheres are co-cultured with a complex mixture containing the target substance (to be separated) because they can specifically bind to the target substance and make it produce a magnetic response. Then the immune microspheres can be selectively bound to the target substance through antigen-antibody reaction. When the compound passes through the magnetic field device, the target substance bound to the immunomagnetic microspheres will be retained by the magnetic field, thus being separated from other compounds.
Immunomagnetic microspheres used for cell separation have the following conditions: stable chemical properties and no coagulation; Non-specific binding with cells; Magnetic microspheres are firmly combined with antibodies; Magnetic microspheres have uniform size, good magnetic response and uniform content of magnetic nano-materials; Magnetic microspheres are suitable in size and are not easily swallowed by cells.
There are two methods to combine IMM with cells, direct method and indirect method. Direct method means that antibodies are directly attached to magnetic microspheres and then combined with target cells. Indirect method refers to the mixed culture of cells and specific antibodies, so that the specific antibodies are bound to the cell surface, and then magnetic microspheres pretreated with anti-mouse IgG (secondary antibody) are added. Magnetic microspheres are indirectly bound to target cells. Direct method can reduce washing and culture steps, but IgM monoclonal antibody is rarely used. Compared with the direct method, the indirect method can use a group of monoclonal antibodies in addition to a wide range of applications, so it will get better cell clearance effect. However, after several washing steps, the specificity will decrease.
The development of monoclonal antibodies against monocytes makes it possible to isolate cells with specific surface markers. Generally, there are three different methods, that is, flow cytometry (PACS), allogeneic red blood cell garland with secondary antibody attached to its surface, and panning technology of passive adsorption antibody on polystyrene tissue culture plate. These technologies all have their own shortcomings. FACS is expensive, complicated in technology and often troubled by the capacity, activity and sterility of selected cells. Red blood cell wreath technology can't handle a large number of cells. In addition, there is no mature and simple method to couple antibodies to erythrocyte membrane. Translation technology also has many limitations. It is difficult to amplify, the steps are complicated and it is impossible to quantify. Target cells are usually mixed with nonspecific antibodies adsorbed on other cells at the bottom of the culture plate. Relatively speaking, immunomagnetic microspheres have the advantages of simple operation, rapid and complete separation, high cell purity, etc., especially in terms of simple operation and time saving, which is incomparable to other separation methods.
Magnetic beads are the key to cell sorting. Take the patented MACS magnetic beads of Miltenyi Biotec Company as an example. The diameter of magnetic beads is only 50 nanometers, which is equivalent to the size of a virus particle and only one millionth of that of eukaryotic cells. Scanning electron microscope can only see the magnetic beads on the cell surface. This kind of magnetic beads can form a stable colloidal solution, which neither precipitates nor polymerizes in the magnetic field. Magnetic beads are composed of iron oxide and polysaccharide, and only bind to one third of the characteristic antigens on the cell surface at most. And because the body is small, it can be degraded by cells, which will not activate or affect the function and vitality of cells, and the physiological functions of cells will not change. Therefore, it is not necessary to remove the magnetic beads after cell separation, and the positively separated cells (that is, magnetically labeled cells) can be used for analysis and subsequent experiments immediately.
MACS technology can separate very pure cell groups with very good recovery and survival rate. According to the difference of cell frequency and antigen marker expression level, the purity of isolated cells can reach 95%-99.9%, and the recovery rate is & gt90%.
The number of cells selected by one-step positive MACS technique can reach 109. Whether the initial unit volume is 105 or 10 1 1, the same simple operation process is used. It takes about 15 minutes to magnetically label cells, and only a few minutes to separate magnetic cells with a sorting column. Columns with different specifications can be used to sort different numbers of cells at will.
Magnetically labeled cells can be stained with fluorescent dyes combined with antibodies at the same time, which can be used for quality control and analysis of cell sorting. Because the magnetic beads of MAC are submicroscopic, they will not affect the scattered light of labeled cells. MACS-sorted cells can be directly measured by flow cytometry, and can also be compatible with fluorescence microscope, PCR or FISH.
MACS technology can be used for positive sorting of rare cells as low as 10-8. For those cells with low frequency, exclusion and positive selection can be combined to separate them. At present, cell separation can also be carried out according to the cytoplasmic protein of cells or the secreted protein of active cells.