Monoclonal antibody technology

Monoclonal antibody technology

Cell fusion technology is used to fuse the immunized B lymphocytes and myeloma cells into hybridoma cells. After screening, after asexual reproduction (cloning) of a single cell, each clone can continue to produce only one epitope. Antibody technology.

Monoclonal antibody is referred to as monoclonal antibody (McAb). Because of its specificity, uniformity, high efficiency and unlimited supply, and the ability to use pure antigens to prepare pure single antibodies, it is used in immunology, medicine, biology, etc. In the field of basic research and clinical medicine, the diagnosis, prevention and treatment of diseases (including cancer) have shown great vitality.

After the vertebrate body is stimulated by foreign antigens, antibodies are produced by the liquid immune system, immunoglobulins, which are distributed in the serum. However, because there are many different epitopes on the surface of the antigen molecule, each epitope can only stimulate the body Corresponding B lymphocytes produce corresponding types of antibodies. An antibody can only bind to its corresponding epitope. Therefore, an antigen-immunized animal with multiple epitopes will have a mixture of multiple antibodies in its serum, so the specificity, homogeneity, and Effectiveness is very low, and production is limited. Such antibody products are difficult to separate and purify even using physical and biochemical means, and therefore cannot meet the needs of medicine and biology for a single pure antibody.

A brief history of development In the early 1960s, it was observed that mouse and rat somatic cells were fused to obtain hybrid cells. In the early 1970s, another mouse myeloma cell line was established. In 1973, C. Milstein and others were studying antibodies The genetic mechanism of the synthesis revealed the "codominantity" of the fused cells, that is, the information from the two parents can be displayed in the offspring; this discovery led G. Keller and C. Milstein to successfully establish the lymph in 1975 Cell hybridoma technology. Since then, scientists from all over the world have used this technology to develop thousands of monoclonal antibodies. At present, commercial production has been achieved.

Basic steps: Cell fusion combines myeloma cells (plasma cell tumor cells) in the logarithmic growth phase of mice or humans (currently multi-purpose mice) with splenocytes of immunized syngeneic animals (spleen is a source of large amounts of B lymphocytes) Mix in the presence of the fusion promoter polyethylene glycol to fuse each other.

Selection of hybridomas The fused cells are divided into selective medium containing HAT (hypoxanthine, aminopterin, and thymidine nuclei) and cultured in 96-well or 24-well tissue culture plates. Because aminopterin can block the main pathway of nucleic acid biosynthesis, and myeloma cells lack hypoxanthine guanine phosphoribosyl transfer (HGPRT) or thymine nuclear stimulation (TK), they cannot use exogenous secondary Xanthine and thymine nuclei are used for nucleic acid synthesis through salvage bypass. Therefore, all hybridomas fused by myeloma cells died. Although splenocytes have these, they can only survive for a few days under in vitro culture conditions. Only hybridoma cells fused by spleen cells and myeloma cells inherit HGPRT and TK secreted by spleen cells The traits, through salvage bypass, use external hypoxanthine and thymine nucleus to synthesize DNA and proliferate.

Screening and cloning of hybridomas that secrete specific antibodies Test the supernatant of each culture well using techniques such as hemagglutination, radioimmunoassay, and joint immunoassay to determine which wells contain hybridoma clones that secrete specific antibodies. hole. Positive wells often contain multiple hybridoma clones, and therefore contain multiple antibodies against different epitopes. Using limited dilution (also known as ten-fold increasing (subtractive) dilution) and other methods can make only one hybridoma in a culture well Cells, this cell reproduces vegetatively to produce a pure cell population (clones). After multiple cloning, each clone can continuously secrete a large number of single high-purity antibodies, called monoclonal antibodies.

McAb preparation and purification in large quantities The McAb content of the hybridoma cell culture fluid is only 10 micrograms per milliliter. If the hybridoma cells are injected into the peritoneal cavity of mice with the same lineage [pre-injected phytan (nor-hhytane)], the content of McAb in ascites produced by the mice can be as high as 5-20 mg per ml. The hybridoma can be frozen and stored in liquid nitrogen, so that it can be recovered at any time to prepare ascites McAb. An immunoaffinity column made of pure antigen can be directly separated from ascites into pure McAb, while high-performance liquid chromatography using hydroxyapatite can prepare a large amount of pure from the supernatant of cell culture fluid or ascites McAb.

Applications in biology Separation and purification, McAb for protein and multi-biological macromolecules can be used to purify a desired substance from the mixture in one step. For example, interferon is a very promising drug for the treatment of viral diseases and cancer, but it is difficult to separate and purify. Using an anti-interferon monoclonal antibody as an immunoadsorption column can purify the interferon 5000 times in one step.

To study the structure and function of proteins, nucleic acids, and gene positioning. McAbs can be used to detect, isolate, and study antigen membrane proteins and hormone receptors of complex biological systems that are difficult to purify; analysis of human serum lipoprotein B antigenic determinants; for human type III Localization in sologens: Recognize different forms of solution and modified forms after translation; Study the genetic variation of alkaline phosphate in placenta; Use protein McAb linked to "hat" to study eukaryotic 5-hat structure (refer to Most of the eukaryotic cell mRNA is added with a molecular structure of a methyl guanine residue at the 5 end after transcription, the latter is a "hat"); using Z-DNA McAb as an immunoadsorption column to separate Z-containing DNA plasmids, etc.

Applications in virology are used for rapid diagnosis, identification, and differentiation of wild strains and vaccine strains; purification of low-concentration viruses that are difficult to isolate; analysis of viral antigen structures; analysis of the molecular basis of viral virulence; study of viral proteins to promote cell fusion Function, and the molecular basis of antigen variation (only one amino acid substitution). In addition, exploratory research has also been conducted on the use of McAb to select mutant strains to prepare influenza virus vaccines.

The application of McAb technology and recombinant DNA technology in biotechnology has been compared. With McAb, you can first determine the specific determinants of the immunogenicity of the infectious agent, and then insert the corresponding gene into the vector, using the in vitro translation system and McAb to detect the gene Is it inserted? Use McAb to find an efficient expression system and detect gene expression products. After gene cloning is efficiently expressed in the expression system, an immunoadsorption column made of McAb can further purify the antigen from complex expression products. The effective combination of McAb technology and genetic engineering has been used to produce biological products such as interferon and subunit vaccines.

It is used in the diagnosis, localization, prevention and treatment of infectious and immune diseases and cancer. McAb is like a "bio-missile" McAb with radioisotopes or McAb with anti-cancer drugs can be directionally combined with cancer cells, the drugs they carry can accurately kill cancer cells, and normal cells are safe and unharmed; Radioisotopes can locate lesions, which is convenient for surgical treatment. Large doses of radioisotopes can also be used for radiotherapy.

McAb can also be used as a standardized reagent for clinical testing for the determination of ABO blood type, pregnancy, hormones and drugs.

In terms of organ transplantation, McAb also has broad prospects. Once a set of McAb of the tested person's tissue matching is commercialized, a matching tissue matching donor can be selected during organ transplantation to ensure the success of the transplantation.

In immunology, McAb has been used to identify and isolate human T cell populations, identify T cell surface antigens, study T cell differentiation and immunoglobulin structure and genetic analysis.

New advances in McAb technology. Preparation of McAb by in vitro immunization. Antigen is added to the suspension of normal mouse spleen cells. After 4 days of in vitro culture, the spleen cells immunized in vitro are taken for fusion or the abdominal cavity of the mouse is cut by anesthesia, and the antigen is injected directly into the spleen Within 80 hours, spleen cells were taken for fusion. The advantage is that the amount of antigen is minimal, the immunization time is short, and the proportion of hybridomas that secrete antibodies is high.

Human McAb produced in vitro cannot be injected directly into the human body due to certain antigens. Human-mouse hybridomas are unstable. Human chromosomes carrying antibody secreting genes are easily lost. McAb from mice can cause harmful immune reactions to the human body. Therefore, scientists have managed to produce human McAbs from outside the body, stimulate human isolated peripheral blood mononuclear cells with antigens, convert these antigen-stimulated cells into lymphoblasts by infectious mononucleosis virus, and then clone them It can continue to grow and secrete the corresponding McAb. It is also possible to fuse transformed lymphoblasts and myeloma cells to prepare McAbs.

Gene engineering technology produces McAb. The antibody gene is inserted into the vector and expressed in the expression system, and then the antibody fragment is taken out to form a functional McAb in the laboratory. A chimera can also be prepared, that is, the variable region that binds to the antigen comes from the mouse and the stable region comes from the human. Such an antibody rarely produces a harmful immune response to humans.

Microcapsule technology produces human McAb. Human-human hybridoma cells grow in porous sugar microcapsules in liquid medium. The small molecules in their metabolites can escape through the microcapsule membrane, while the cells themselves and McAb remain inside. The microcapsules are separated from the culture liquid, and after washing and crushing, a high concentration of McAb can be obtained.

The hollow fiber bioreactor can be used to cultivate hybridoma cells on a large scale, and can produce 20-40 grams of McAb per month. The selective electrofusion technology mediated by biotin-avidin enables the hybridomas produced to secrete specific antibodies with high affinity, which greatly shortens the screening process.

McAb's anti-idiotype antibody will be a new way to prepare a complete vaccine