The basic principle of hybridoma antibody technology is to fuse both cells while maintaining the main features of both. These two cells are antigen-immunized mouse B cells and mouse myeloma cells, respectively. The main feature of spleen lymphocytes is its antibody secretion function and its ability to grow in selective medium (see the selection principle). Mouse myeloma cells can divide and proliferate indefinitely under culture conditions, so-called immortality. Under the action of the selection medium, only the hybridization of B cells and myeloma cells has the ability to proliferate continuously, forming a cell clone that has both the secretory function of the antibody and the immortality of the cell. The principle is clarified from the following three main steps.
(1) Selection and fusion of cells The purpose of establishing hybridoma technology is to prepare monoclonal antibodies specific for antigens. Therefore, one of the fusion cells must select B cells that are immunized with antigen, usually derived from spleen cells of immunized animals. The spleen is an important place for B cell aggregation. No matter what kind of immune stimulation, there will be obvious antibody response in the spleen. The other side of the fused 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 cell fusion agent is used to cause a certain degree of damage to the cell membrane, so that the cells are easily adhered to each other and fused together. The fusion effect of zui Jia should be a low degree of cell damage in Zui and a high frequency fusion of Zui. Polyethylene glycol (PEG1000～2000) is a commonly used cell fusion agent in Zui, and the concentration is generally 40% (W/V).
(B) Selection of medium application 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 to 7 days in the culture medium without special screening; the multimeric form of the cells is also prone to die; whereas 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 synthesize DNA. Folic acid is an important coenzyme involved in this synthesis process. Another 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 For 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 antigen 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 plurality 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 substantial proportion of unrelated cell fusions in cells that have been fused 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.
Proteins are large biomolecules, or macromolecules, consisting of one or more long chains of amino acid residues. Proteins perform a vast array of functions within living organisms, including DNA replication, catalyzing metabolic reactions, responding to stimuli, and transporting molecules from one location to another.
We supply protein, which is generally consumed immediately before and after exercising, or in place of a meal. Some types of protein are to be taken directly before and after a workout, while others are to be taken before going to bed. The theory behind this supplementation is that bodybuilders, by virtue of their unique training methods and end-goals, require higher-than-average quantities of protein to support maximal muscle growth.
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