What is a monoclonal antibody? What are its characteristics and application value?

Classification: molecular biology knowledge number: large, medium and small 1975 Kohler and milstein first reported that spleen cells of mice were immunized with sheep red blood cells (SRBC) and fused with myeloma cells of mice by cell hybridization technology, thus creating the first B-cell hybridoma cell strain and obtaining monoclonal antibodies against SRBC. They pioneered the fusion technology of B lymphocytes secreting specific antibodies and infinitely proliferating myeloma cells, which laid the foundation for the preparation of monoclonal antibodies. This is a milestone in the history of immunology and even medicine.

Monoclonal antibodies refer to homologous antibodies produced by monoclonal hybridoma cells that only recognize specific epitopes.

Monoclonal antibody is a kind of special antibody prepared artificially, which has the properties of general antibody. It is a globulin produced by the proliferation and differentiation of B cells into plasma cells, which exists in body fluids and has immune function and mediates humoral immunity. It can specifically bind with corresponding antigens (such as pathogens) and play an immune role with the participation of other immune molecules and cells.

The essence of monoclonal antibody is globulin, which has physical and chemical properties, spatial structure and biological activity. Globulin is sensitive to heat and chemicals, and its structure is easily destroyed by heating and adding chemical reagents, so it has a certain half-life. Like globulin, there are variable regions and constant regions, and the variable regions combine with antigens to form antigen-antibody complexes. There is a complement binding site in the constant region, which mediates the complement to play a role, forms a membrane-attacking complex and kills mutant cells.

Monoclonal antibodies have both the general properties of antibodies and their particularity. It is highly consistent in structure and composition, and its type antigen binding specificity and affinity are completely the same. It is easy to be prepared and purified in large quantities in vitro, and can be widely used in the fields of medicine and microbiology. Monoclonal antibodies fuse mouse immune spleen cells (B cells) with infinitely proliferating myeloma cells to form hybridoma cells. This hybridoma cell not only has the ability of infinite reproduction, but also has the ability of secreting antibodies, so it is in vitro.

The preparation of monoclonal antibodies is complex, involving a variety of biotechnology. This process is briefly described here. Pure strain animals (such as Bala-C mice) are immunized with specific antigens, and repeated subcutaneous injections are made in the back. Each injection should be quantitative, and the inoculation interval should be paid attention to, and Freund's adjuvant can be added appropriately. After feeding for a period of time, the inoculated animals with high serum antibody were killed under aseptic conditions, and the spleen was taken out and separated. Centrifuge the suspended cells and put them in a Petri dish for later use. The myeloma cells of pure mice were put into a Petri dish, and some feeder cells (such as thymocytes and macrophages) were added to promote their growth. There are many kinds of cells in the mixed solution. In order to extract target cells-hybridoma cells, HAT selection must be carried out to obtain pure hybridoma cells. In many hybridoma cells, some do not express immunoglobulin, while others express other types of globulin. Therefore, positive hybridoma cells should be screened from many cells by specific detection methods. The target cells should be separated into single cells and cloned into cell strains, and then transferred to other media for expanded culture. In order to obtain a large number of antibodies, it is necessary to inoculate mice intraperitoneally, and then isolate and purify antibodies from ascites.

Isolation, purification and detection of monoclonal antibodies. Put the culture medium amplified in vitro or ascites inoculated in mouse abdominal cavity into a centrifuge tube for high-speed centrifugation, take its supernatant and put it on a glass slide, and add the corresponding antigen to see if there is coagulation.

After the advent of monoclonal antibodies, brilliant achievements have been made, such as the treatment of Corynebacterium diphtheriae with monoclonal antibodies against diphtheria exotoxin; Monoclonal antibody against lipopolysaccharide A is used to treat G~ (2+) septicemia. Monoclonal technology won the Nobel Prize in Medicine 65438-0984. Its main functions are: using monoclonal antibodies instead of polyclonal antibodies to overcome cross-reactions, improve the specificity and sensitivity of immunological experiments, and thus promote the development of medical laboratory science; Using monoclonal as affinity column, soluble antigens with extremely low content, such as hormones, cytokines, tumor antigens and other antigens that are difficult to purify, can be separated and purified. It opens up a new way for material purification; The prepared monoclonal antibody recognizes cell surface specific receptors, and if anti-tumor drugs (such as toxins or radioactive substances) are coupled to it to construct biological missiles, it is expected to overcome human diseases-tumors.

Monoclonal antibody has a broad development prospect, but it also has its limitations. Monoclonal antibody, as an exogenous antigen, can induce immune response, produce corresponding anti-antibody and produce rejection after repeated injection. Therefore, the clinical use of monoclonal antibodies is risky and can be used with caution in case of emergency prevention.

In a word, monoclonal antibody has made a great breakthrough for human beings in detecting and treating diseases, although it still has some shortcomings. I believe that in the near future, human beings will prepare more excellent monoclonal antibodies and make greater contributions to overcoming tumors!

Monoclonal antibodies have the characteristics of highly consistent physical and chemical properties, single biological activity, strong binding specificity with antigens, convenient manual operation and quality control, and easy source. These advantages make it highly valued as soon as it comes out and widely used in biology and medical research.

1 as affinity chromatography ligand

Monoclonal antibodies can specifically bind to their corresponding antigens, so they can identify individual components from complex systems. As long as the monoclonal antibody against a certain component is obtained, it can be fixed on a chromatographic column as a ligand, and this specific component can be separated and purified from a complex mixture by affinity chromatography. If anti-human chorionic gonadotropin (hCG) affinity chromatography column is used, pure hCG can be extracted from pregnant women's urine. Compared with other extraction methods (precipitation method, high performance hydrophobic chromatography, etc.). ), which has the advantages of simplicity, rapidity, economy and high product activity.

As a guiding weapon for biotherapy.

Liposomes are continuous double-layer microcapsules composed of hydrophilic and lipophilic amphiphilic phospholipids, which contain water-phase space and can wrap water-soluble substances. Liposomes coated with cytotoxic drugs are conjugated with antibodies, which can attack target cells directionally, and are called immunoliposomes. This "guided therapy" has achieved satisfactory results in both animal experiments and in vitro experiments. For example, heat-sensitive immunoliposomes are anti-human breast cancer cell antibodies modified with hydrophobic long-chain fatty acids. The antibodies have long hydrophobic carbon chains, which are partially inserted into the lipid bilayer membrane of liposomes, and the Fab segment of antibodies is still exposed on the membrane surface, thus maintaining the antibody activity. Thermosensitive immunoliposomes can specifically recognize the target cells (human breast cancer cells), and rupture the liposomes through the phase transition temperature, thus directionally releasing drugs. In addition, cytotoxic agents such as chemotherapeutic drugs, bacterial toxins, phytotoxins or radioisotopes can be directly cross-linked with monoclonal antibodies against anti-tumor antigens, and cytotoxic agents can be located in tumor cells and directly kill tumor cells by their targeting effect. This not only improves the curative effect of antibody, but also reduces the toxic reaction of cytotoxic agent to normal cells. If anti-T cell monoclonal antibody and daunorubicin are used in combination, there is no killing effect on non-T lymphocytes in vitro. However, there are still many technical difficulties in applying this method to clinic, including the rejection of mouse monoclonal antibodies by human body.

3 as an immunosuppressant

As a new immunosuppressant, monoclonal antibody against human T lymphocytes has been widely used in the clinical treatment of autoimmune diseases and organ transplant rejection. Its mechanism of action depends on the types of monoclonal antibodies and their immunological characteristics. Injection of monoclonal antibody against mouse Thy- 1 antigen can inhibit skin transplantation rejection in mice. In addition, donor bone marrow for allogeneic bone marrow transplantation can be treated with anti-T cell monoclonal antibody and complement in vitro, which can reduce the occurrence of graft-versus-host disease.

As a probe in research work.

Monoclonal antibodies only bind to an epitope (antigenic determinant) on antigen molecules, so they can be used as probes in research. At this time, we can study the relationship between the structure and function of antigen at different levels of molecules, cells and organs, and then clarify its mechanism in theory. If monoclonal antibodies labeled with fluorescent substances are used as probes, the position and distribution of corresponding biological macromolecules (protein, nucleic acids, enzymes, etc.). ) can be easily determined in cells.

5. Enhance the immunogenicity of antigen.

Antibodies have a long history of enhancing the immunogenicity of antigens. In 1960s, it was found that it was difficult for piglets to produce tetanus toxoid antibodies. Injecting the corresponding specific antibody IgG can effectively improve the immune response to Venezuelan equine encephalitis virus. Since 1984, Celis et al. have found that anti-HBs)IgG can enhance the proliferation of specific human T cell clones stimulated by HBs antigen and induce interferon. In mice, it was found that when the low dose of HBs antigen did not produce immune response, adding a complex composed of anti-HBs antibody could effectively induce immune response. According to this function, a therapeutic vaccine of hepatitis B antigen-antibody complex was developed.

6 as a medical test reagent

As a medical test reagent, monoclonal antibody can give full play to its advantages. Monoclonal antibody has strong specificity, which greatly improves the specificity of antigen-antibody reaction, reduces the possibility of cross-reaction with other substances, and makes the test results more reliable. The homogeneity of monoclonal antibodies and the singleness of biological activity make the results of antigen-antibody region easy to control and standardize. At present, many detection kits are made of monoclonal antibodies, and their main uses are as follows:

(1) Diagnosing various pathogens

This is the most widely used field of monoclonal antibodies, and there are a large number of diagnostic reagents to choose from. For example, reagents for diagnosing hepatitis B virus, hepatitis C virus, herpes virus, cytomegalovirus, EB virus and various microbial and parasitic infections. Because of its high sensitivity and specificity, monoclonal antibodies have more unique advantages in identifying strains and subtypes, virus variants and antigenicity of parasites in different life cycles.

(2) Detection of tumor-specific antigen and tumor-associated antigen

It is used for the diagnosis, classification and localization of tumors. Although monoclonal antibodies against tumor-specific antigens have not been prepared, monoclonal antibodies against tumor-related antigens (such as alpha-fetoprotein, tumor basic protein and carcinoembryonic antigen) have long been used in clinical trials.

With the application of lymphocyte hybridoma technology, many hybridoma cell lines with anti-human tumor markers have been established, which laid a foundation for early diagnosis of tumors, elucidation of the occurrence and development of tumors, understanding of the biological activity of tumor cells and quantitative research. Examining pathological specimens with anti-tumor monoclonal antibodies is helpful to determine the primary site of metastatic tumors. Monoclonal antibody labeled with radionuclide can be used for in vivo diagnosis, and combined with X-ray tomography, it can quantitatively diagnose the size and metastasis of tumor.

(3) Detection of lymphocyte surface markers

Used to distinguish cell subsets and differentiation stages of cells. For example, the detection of CD series markers is helpful to understand the changes of cell differentiation and the quantity and quality of T cell subsets, which is of reference significance for the diagnosis of many diseases. The detection of cell surface antigen will play a guiding role in the disease stage, treatment effect and prognosis judgment of leukemia patients. Detection of histocompatibility antigen is an important content of transplantation immunology, and more reliable results can be obtained by detecting its site with monoclonal antibody.

(4) Determination of trace components in organisms.

Using monoclonal antibodies combined with other technologies, a variety of trace components in human body can be determined. For example, radioimmunoassay is a method established by using the sensitivity of isotopes and the specificity of antigen-antibody reaction. It can measure10-9 ~10-12g, so that hormones that were difficult to measure can be quantitatively analyzed. In addition to hormones, many biochemical substances such as enzymes, vitamins and drugs can be detected. This is of practical significance to the health status judgment, disease detection, guiding diagnosis and clinical treatment of the subjects.

To sum up, the application of monoclonal antibodies in theory and practice has become an important means to solve many important problems in biology and medicine. However, the monoclonal antibody used above belongs to mouse origin, and it will produce heterogeneous protein allergy when used for the prevention and treatment of human diseases, which greatly limits its clinical application value. Moreover, the half-life of mouse antibody in human body is shortened and its biological activity is reduced. Therefore, people have been committed to the study of human-derived antibodies, such as the hybridization between human spleen cells and mouse myeloma cells; Transforming human B lymphocytes with EB virus; Preparation of chimeric antibody by genetic engineering. But they all met with considerable difficulties. It is the latest method in recent years to extract and amplify DNA encoding human antibody, construct plasmid, establish combinatorial antibody library, and then use donor library to establish sub-library for a specific antigen. The antibody fragment (Fab) prepared by this technique is a human antibody, which has the characteristics of simple library building and stable antibody expression. So far, a variety of antibodies have been produced. It can be predicted that this technology has a good development and application prospect.

Review of Monoclonal Antibodies

First, the definition of antibody and the first generation of monoclonal antibody (produced by hybridoma cells)

Antibody is a glycoprotein produced by B lymphocytes in the immune response to antigen stimulation. It is a kind of globulin, which can specifically bind with the corresponding antigen and produce various immune effects (physiological effects). The International Health Organization named a class of protein with antibody activity and similar chemical structure to immunoglobulin as immunoglobulin, which refers to the protein with the same antibody. Between 70000 and 70000, it is called "heavy chain" (H chain). Light chain, heavy chain and double chain are connected by disulfide bonds, and such a four-chain structure (L2H2) constitutes immunoglobulin molecules. 550 amino acid residues with a relative molecular weight of 55,000; The basic structure of the antibody is shown in the figure, which consists of four peptide chains, of which two peptide chains with relatively low molecular weight contain about 2 10 amino acid residues, and the relative molecular weight is about 24,000, which is called "light chain" (L chain); The other two peptide chains with relatively large molecular weight contain about 450, and their corresponding antibodies are named IgG, IgM, IgA, IgE and IgD respectively. Serological classification of antibodies is based on antigenic differences of heavy chains: animals are immunized with various separated and purified heavy chains to obtain corresponding antiserum, and then the similarities and differences of their structures are analyzed by serological detection methods such as immune cross reaction. After repeated verification, it was finally found that human beings have five different types of heavy chains, namely, (1) binding with various cells, and (5) possibly causing immune damage. IgG is the main antibody in human serum, accounting for more than 75% of the total antibodies in adult serum. It is produced by B cells, and its functional structure is also the most clearly studied. Its main physiological functions are: (1) neutralizing toxins and viruses, (2) agglutinating and precipitating antigens, (3) activating complement, and (4) using specific membrane receptor (Fc) monoclonal antibody technology is one of the important advances in the field of modern biological science. Monoclonal antibody has a wide range of application values, which opens up a new way for the research of natural sciences such as biology and medicine.

The second generation monoclonal antibody: chimeric or humanized monoclonal antibody after gene recombination.

Chimeric antibody: refers to replacing the constant region of mouse with the constant region of adult and retaining the variable region sequence of mouse monoclonal antibody to form human-mouse hybrid antibody. Its development procedure is fast, which can greatly reduce the immunogenicity of heterologous antibodies, but almost retains all the specificity and affinity of parent mouse monoclonal antibodies. In addition, it also has the function of human antibody, such as complement binding, antibody-dependent cell-mediated cytotoxicity (ADCC) and so on. Examples of successful chimeric antibodies are:

Rituximab: mouse anti-CD20 antibody of IDEC Pharmaceutical/Genentech, containing human IgG 1κ constant region, used to treat B lymphoma. Its anti-lymphoma effect may mainly come from complement action, ADCC and inducing tumor cell apoptosis.

Remicade: anti-TNF-α antibody of Centocor Company, used to treat rheumatoid arthritis and Crohn's disease.

Imitation: anti-CD25 antibody of Novartis, which is used to resist transplant rejection.

Ebitur: An anti-EGFR(Her- 1) monoclonal antibody from Imclone Company of the United States has been approved by FDA for the treatment of colorectal cancer.

Humanized antibody: Using numerous existing mouse antibodies that have been analyzed in detail, the antibody fragment (CDR) that is in direct contact with the antigen was grafted with the human antibody framework. After affinity modification, it can maintain its specificity and most of its affinity, while almost removing its immunogenicity and side effects.

Successful examples:

Herceptin: An anti-HER2/neu antibody from Genentech, used to treat breast cancer.

Synsgis: anti-F antibody of Medimmune Company, used to treat respiratory virus infection.

Zenapax: Protein Design Laboratory (PDL)/ Roche anti-CD25 antibody, which is used to resist transplant rejection.

H-R3: Anti-EGFR(Her- 1) monoclonal antibody of Cuban Center for Molecular Immunology, used to treat head and neck cancer.

Three, phage display antibody and whole human antibody

Preparation of human antibodies by (1) phage display technology

Based on the ability of phage to express antibody fragments on the outer membrane, a series of antibody libraries were established, and then the corresponding antibody fragments in the libraries were screened with target antigens, and then functional complete human antibodies could be formed by in vitro processing. Monoantibodies against simple or complex antigens can be prepared by this method, and antibodies with moderate affinity can be obtained, but this method needs to be screened by Qualcomm quantity. There are some clinical phase II/III studies, such as D2E7 and CAT- 152 of Cambridge antibody technology, which are used to treat rheumatoid arthritis and glaucoma respectively. D2E7 is an anti-TNF-α monoclonal antibody developed by Abbott Laboratories. Judging from the clinical phase III data provided in June 2002, it is expected to enter the market in the near future.

(2) Transgenic animals prepare human antibodies.

Antibody derivatives: Some researchers are developing monoclonal antibody derivatives that are easier to produce and use, the most successful of which is Mark Green of the University of Pennsylvania. He was looking for a small polypeptide molecule [16] derived from CDR of antibody complementarity determining region, and established CDR therapy (now belonging to Xcyte Therapeutics) to accelerate the commercialization of its polypeptide production. His polypeptide has the affinity and selectivity of parent monoclonal antibody, but as a small molecule, it should be non-immunogenic and easier to prepare.