SPG-(Streptococcal Protein G) antibody polymer and preparation method as well as application thereof

Abstract

The invention discloses SPG-(Streptococcal Protein G) antibody polymer and a preparation method as well as application thereof. The SPG-antibody polymer is formed by coupling anti-glycophorin A monoclonal antibody, an SPG antibody and a target antigen antibody. One end of the polymer is coupled with red blood cells through the anti-glycophorin A monoclonal antibody, and the other end of the polymer is coupled with a target antigen through antibodies of a surface antigen of a target cell and other antigens; then standing is performed to detect the target antigen through red blood cell precipitation, or mature nucleated cells and red cells in blood are removed through density gradient centrifugation; and all stem cells/progenitor cells are reserved. In the invention, a combined method of negative selection and density gradient centrifugation is adopted for separating and extracting the stem cells and the progenitor cells, and markers are not available on the surfaces of the collected progenitor/stem cells, so that the antibody polymer has the advantages of high cell activity, simple and practical method, industrial production and easiness in popularization, can be widely applied to research and treatment of laboratory and clinical cell damage diseases and has broad prospect.

Description

technical field [0001] The invention belongs to the application field of biomedical technology, and in particular relates to an SPG-antibody multimer and its preparation method and application. SPG-antibody multimer is the abbreviation of anti-glycophorin A monoclonal antibody-streptococcal G protein-antibody multimer. Streptococcal protein G is abbreviated as SPG. Background technique [0002] Stem cells (stem cells, SC) are a type of cell population with self-renewal, high proliferation and multi-directional differentiation potential, that is, these cells can maintain the size of their own cell population through cell division, and at the same time can further differentiate into various tissues Cells make up various complex tissues and organs of the body. Stem cell therapy technology, also known as regenerative medical technology, refers to the treatment of diseases through the isolation of stem cells and targeted transplantation into damaged tissues or organs. [0003]...

AI Technical Summary

Problems solved by technology

[0004] At present, the separation of stem cells from human blood mainly includes flow cytometry, immunomagnetic bead method, blood cell separator, in vitro culture, etc. The problems of these methods are: first, the specific markers on the surface of the isolated stem cells are not conducive to clinical application; Second, only one type of stem cells can be isolated, and all types of stem cells cannot be retained; third, the cost of separation is high; fourth, the contamination rate of in vitro cultured cells is high, the plasticity of cells is weakened, and the function is poor

[0005] There are methods (Chinese patent application 200910187212.4 and Chinese patent application 200610106875.5) to directly separate stem cells with methylcellulose or hydroxyethyl starch and polysucrose-diatrizoate meglumine separation solution (Ficoll reagent) with a density of 1.074-1.077g / ml, Although this method can remove plasma, platelets, plasma proteins and most red blood cells, what is obtained is all nucleated cells, namely mature monocytes, lymphocytes, multinuclear cells, granulocytes and all stem cells, progenitor cells, etc. , stem cells cannot be obtained directly, and the purity of stem cells in the obtained cells is poor and there are residual red blood cells

Experiments and numerous literatures have shown that the stem cell yield of cells separated by the appeal method is generally not higher than 70%, and when umbilical cord blood is used as the source of stem cells, the resulting cells contain a large number of lymphocytes, NK cells, granulocytes, stem cells The purity is extremely low, and it is easy to cause graft-versus-host and other immune reactions after transplantation, and some residual red blood cells are easy to cause hemolysis, which brings huge safety risks to the clinic

For this reason, in the Chinese patent application 200710137781.9, this problem is improved, and a kit for in vitro separation of bone marrow and cord blood stem cells and its application method are disclosed. cells, but there are still these problems: First, in this patent, the Lin antibody is attached to macromolecular hydroxyethyl starch or methylcellulose, which involves the chemical synthesis of macromolecules and product purification. The reaction between the two is difficult, and the reaction products are difficult to control. , the steps are cumbersome, and the final yield is extremely low, causing a lot of waste of antibodies and greatly increasing the cost

Secondly, the specific antibody composition and concentration of the Lin antibody in this patent are not given, and the type, purity, and cell quality of the finally separated cells cannot be guaranteed; finally, in this embodiment of the patent, the antibody is All the components of the kit are sterilized under high pressure at 121°C for 35 minutes, which makes all antibody proteins inactivated due to denaturation, which leads to problems in the implementation of this patent

In Chinese patent application 201010022753.4, the SPA-antibody trimer, the cell treatment kit containing the trimer and its preparation method and application are disclosed. This technical solution can process peripheral blood / bone marrow / umbilical cord blood or other Animal blood can be separated to obtain nucleated cells or detect antigens, but there are still these shortcomings: the binding spectrum of SPA and immunoglobulin is narrow, and the binding is weak

Secondly, the cd3, cd19, cd14, and cd56 monoclonal antibodies used in this patent for cell processing kits to separate stem cells from peripheral blood / bone marrow blood / umbilical cord blood can respectively label and remove most of the T lymphocytes and B lymphocytes in the blood And monocytes and NK cells, can not remove a large number of granulocytes, macrophages

The residual lymphocytes, monocytes, NK cells and a large number of granulocytes and macrophages in the isolated stem cells pose a huge safety hazard to its clinical application