Major applications: hybridoma technology

Hybrid hybridoma refers to a phenomenon in which two or more cells combine to form a single cell. It can make the nucleus of two different sources express function in the same cell. Fusion of myeloma cells and immune lymphocytes (immunized B lymphocytes) forms hybridoma cells capable of secreting highly pure monoclonal antibodies.

 

Hybridoma technique

Lymphocyte hybridoma technology, also known as monoclonal antibody technology, is based on somatic cell fusion technology. Kohler and Milstein demonstrated that myeloma cells fuse with spleen cells of immunized animals to form a highly specific antibody that secretes high specificity against the antigen, a monoclonal antibody. The basis of this technology is cell fusion technology. Myeloma cells can be serially passaged in vitro, while spleen cells are terminal cells and cannot be propagated in vitro. For example, when a mouse myeloma cell is fused with a lymphocyte secreting an antibody or a factor, the fused cell has both the infinite reproductive characteristics of the tumor cell and the ability of the lymphocyte to secrete specific antibodies or factors, and overcomes the problem. The disadvantage is that immune lymphocytes cannot be propagated in vitro, and the fused cells are called lymphocyte hybridomas.

 

Principles and steps of hybridoma:

The basic principle of hybridoma technology is to fuse both cells while maintaining the main features of both. These two cells are antigen-immunized mouse spleen cells and mouse myeloma cells, respectively. The main feature of mouse spleen cells (B lymphocytes) immunized by specific antigens is its antibody secretion function, but it cannot be continuously cultured in vitro, and mouse myeloma cells can divide and proliferate indefinitely under culture conditions, that is, the so-called immortality. Under the action of the selective medium, only the hybrid cells in which the B cells are fused with the myeloma cells can have the ability to continue to culture, and form a cell clone having both the secretory function of the antibody and the immortality of the cells.

The principle is clarified from the following three main steps:

(1) Cell selection and fusion

The purpose of establishing hybridoma technology is to prepare monoclonal antibodies specific for the antigen, so the fusion cell must select B cells that are immunized with the antigen, usually derived from the spleen cells of the immunized animal. The spleen is an important place for B cell aggregation. No matter what kind of immune stimulation, there will be obvious antibody responses in the spleen. The other side of the fusion cell is to maintain the proliferation of cells after cell fusion, and only tumor cells have this characteristic. Selecting cells from the same system increases the success rate of fusion. Multiple myeloma is a malignant tumor of the B cell line, so it is an ideal spleen cell fusion partner.

The use of cell fusion agents causes a certain degree of damage to the cell membrane, allowing cells to adhere to each other and fuse together. The best fusion effect should be minimal cell damage with the highest frequency of fusion. Polyethylene glycol (PEG 1 000~2 000) is currently the most commonly used cell fusion agent, and the general application concentration is 40% (W/V).

(2) Application of selective medium

Cell fusion is a random physics process. In a mixed cell suspension of mouse spleen cells and mouse myeloma cells, the cells will appear in various forms after fusion, such as fused splenocytes and tumor cells, fused splenocytes and spleen cells, fused tumor cells and tumor cells, unfused spleen cells, unfused tumor cells, and multimeric forms of cells, and the like. Normal spleen cells survive only 5-7 days in the culture medium, and no special screening is required; the multimeric form of the cells is also easy to die; and the unfused tumor cells require special screening and removal.

There are generally two ways to synthesize cellular DNA. The main route is to synthesize nucleotides from sugars and amino acids, and then to synthesize DNA. Folic acid is an important coenzyme involved in this synthesis process. The secondary route is the synthesis of DNA by the catalytic action of hypoxanthine phosphoribosyltransferase (HGPRT) and thymidine kinase (TK) in the presence of hypoxanthine and thymidine. There are three key components in the cell fusion selection medium, hypoxanthine (H), aminopterin (A) and thymidine (T), so the three heads are called HAT medium. Methotrexate is an antagonist of folic acid, which blocks tumor cells from synthesizing DNA by normal routes, and the tumor cells used for fusion are HGPRT-cell strains selected by toxic medium, so they cannot grow in this medium. Only the fused cells have the genetic properties of both parents, and can survive and multiply in HAT medium for a long time.

(3) Limited dilution and antigen-specific selection

In animal immunization, high purity antigens should be used. An antigen often has multiple determinants. The humoral immune response produced by an animal after being stimulated by an antigen is essentially the secretion of antibodies from a large number of B cell populations, while the B cells targeting the target antigenic epitope are only a small fraction. Since cell fusion is a random process, a considerable proportion of unrelated cell fusions in the already fused cells are screened for removal. The screening process is generally divided into two steps: one is antibody screening of fused cells, and the other is specific antibody screening based on this. The fused cells are fully diluted so that the number of cells allocated to each well of the culture plate is between 0 and several cells (30% of the wells are 0 to ensure a single cell in each well). The supernatant was subjected to ELISA to select highly secreting cells of the antibody; this process is often referred to as cloning. These positive cells were further cloned, and an antibody-positive cell line against the target antigen was identified by using an ELISA coated with a specific antigen, and then subjected to cryopreservation, in vitro culture, or intraperitoneal inoculation culture.

Hybridoma technology application:

Monoclonal antibodies are of great value not only in basic research in cell biology and cdc immunology, but also in combination with the application of gene therapy, and are widely used in practice. In medicine, monoclonal antibodies have been used in the diagnosis of diseases, and their advantages are accurate diagnosis and no cross-reactivity. For example, when monoclonal antibodies are used to diagnose hepatitis B and latent hepatitis B virus, false negatives are rarely missed. Monoclonal antibodies can also be used as a pharmaceutical carrier for the treatment of diseases. Monoclonal antibodies have specific affinity for target tissues, so they have specific localization and distribution characteristics in vivo. Combining anti-tumor drugs with monoclonal antibodies against certain tumors allows the drug to selectively concentrate on the tumor cells in the body, killing only the target cells without damaging the normal tissues, and greatly reducing the anticancer drugs. side effect. Therefore, drug-loaded monoclonal antibodies are known as “biological missiles.” Most of the monoclonal antibodies currently produced are mouse-mouse type, which is a heterologous protein for humans and therefore difficult to treat. In order to solve the human body’s rejection of heterologous monoclonal antibody proteins, scholars are working hard to develop human-human monoclonal antibodies, which are beneficial for the treatment of diseases.