According to the principles and methods of preparation, antibody preparation services can be divided into three categories: polyclonal antibodies, monoclonal antibodies, and genetically engineered antibodies.
Polyclonal antibody preparation: Medicilon polyclonal antibody preparation service has professional antibody preparation personnel, advanced antibody preparation and production equipment, and the introduction of advanced preparation technology. Medicilon professionally creates preparation solutions according to customer needs. The main animal options provided include: Rabbits, mice, rats, etc.
Monoclonal antibody preparation: Medicilon monoclonal antibody preparation service content (1) Immunization and ELISA detection: customers provide protein or peptide coupled with carrier protein (such as KLH), need about 4mg protein immunosource or 5~10mg peptide, immune 5 For Balb/c mice, blood was collected and tested by ELISA. (2) Fusion and Shuaixuan: Mouse spleen cells are fused with myeloma cells, and the resulting hybridoma cells are cloned by proliferation and ELISA screening to obtain high valency. (3) Subcloning and delivery results: The two best clones are selected for subcloning and subtype identification. The delivery results include a flask of 5ml culture supernatant per clone.
Preparation of genetically engineered antibodies: Successfully studied genetically engineered antibodies include: chimeric antibodies, reconstituted antibodies, and small molecule antibodies.
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Most natural antigen substances (such as bacteria or their exotoxins and various tissue components) often have many different antigenic determinants, and each determinant can stimulate the body to produce a specific antibody.
Polyclonal antibody is a mixture of multiple antibodies secreted by multiple immune lymphocytes after multiple antigenic determinants stimulate the body.
Because this antibody is heterogeneous, whether it is the study of antibody molecular structure and function or the clinical application are greatly restricted, it is called the first generation antibody;
Thousands of antibody-forming cells (ie B cells) can exist in the body’s lymphoid tissue. Each antibody-forming cell only recognizes its corresponding antigenic determinant. When stimulated by an antigen, it can proliferate and differentiate into a cell population. A cell population formed by single cell proliferation may be referred to as a clone.
B cells of the same clone can synthesize and secrete homogenous antibodies that are identical in physical and chemical properties, molecular structure, genetic markers, and biological characteristics, and can also be called monoclonal antibodies. Monoclonal antibodies are high-purity antibodies that only target a specific antigenic determinant, so they are called second-generation antibodies.
1. Advantages
(1) Pure antibody can be obtained without purification of antigen during preparation;
(2) has high titer
(3) High degree of unity and uniformity;
(4) High output and continuous production
2. Basic principles of monoclonal antibody preparation
It is difficult to obtain monoclonal antibodies by in vivo immunization. If the required antibody-forming cells can be selected and cultured in vitro, known specific monoclonal antibodies can be obtained.
Hybridoma cells: not only have the characteristics of myeloma cells capable of unlimited growth and reproduction, but also have the ability of antibody forming cells to synthesize and secrete antibodies. This antibody is a homogeneous antibody produced by a cell clone that recognizes an epitope, so it is called a monoclonal antibody.
Application of hybridoma technology can obtain monoclonal antibodies for almost all antigens, as long as such antigens can elicit antibody responses in mice.
(1) DNA synthesis
① Main way: Refer to biochemistry, which can be blocked by aminopterin (A)
② Alternative pathway: use hypoxanthine (H) and thymidine (T) to synthesize DNA;
The required enzymes are hypoxanthine-guanine phosphoribosyl transferase (HGPRT) and thymidine kinase (TK).
(2) Myeloma cells: myeloma cell lines derived from BABC/c mice; no ability to synthesize antibodies; ability to grow and reproduce indefinitely in vitro; loss of ability to synthesize HGPRT and TK; DNA can only be synthesized by main routes; It cannot grow in HAT medium containing hypoxanthine (H), aminopterin (A), and thymidine (T).
(3) Immune lymphocytes: usually derived from splenic lymphocytes of BALB/c mice; capable of synthesizing antibodies; capable of synthesizing HGPRT and TK; capable of synthesizing DNA through main and alternative pathways; capable in HAT medium Growing, but short-lived, can only survive for a few days, does not have the ability to grow and reproduce in vitro
(4) Fusion agent: Kohler and Milstein’s earliest Sendai virus; later found that polyethylene glycol (PEG) fusion effect is better, and avoid the problem of virus pollution.
(5) Hybridoma cells: have the ability to synthesize antibodies (obtained from immune lymphocytes); have the ability to synthesize HGPRT and TK (obtained from immune lymphocytes); can synthesize DNA through main and alternative pathways; in HAT medium Can grow; in vitro has the ability to grow and reproduce indefinitely (obtained from myeloma cells).
(6) Feeder cells: Single or a few cells are difficult to survive in vitro culture, and other cells must be added to increase cell density, maintain their metabolism, and make them survive and reproduce; the added cells are called “feeder cells”. The most commonly used mouse peritoneal macrophages.
3. Preparation process of monoclonal antibody:
Preparation of antigens → Immunization of animals → Preparation of immune spleen cells and myeloma cells → Cell fusion → Selection and culture of hybridoma cells → Screening of hybridoma cells
→ Cloning of hybridoma cells → Verification of monoclonal antibodies → Establishment of hybridoma cell lines → Mass production of monoclonal antibodies (animal in vivo and in vitro culture methods)
4. Application of monoclonal antibodies
(1) Diagnostic reagents: used for the diagnosis of various pathogens and tumors.
(2) Therapeutic reagents: treatment of infectious diseases in veterinary clinics; suppression of organ transplant rejection in clinics, treatment of autoimmune diseases, and preparation of biological missiles.
(3) Detection reagents: detection of virulence factors of various pathogens; detection of surface molecules of various immune cells and other tissue cells.
Since the advent of monoclonal antibody hybridoma technology in 1975, monoclonal antibodies have been widely used in the diagnosis and treatment of diseases in medicine. However, at present, most of the monoclonal antibodies are of murine origin, and anti-mouse antibodies are produced in the body during repeated clinical administration, which weakens or disappears the clinical efficacy. Therefore, the most ideal monoclonal antibody in the medical clinic should be human, but the human-human hybridoma technology has not yet been breakthrough.
The current better solution is to develop genetically engineered antibodies to replace mouse monoclonal antibodies for clinical use. Genetically engineered antibodies emerged in the early 1980s. They use DNA recombination technology to cut, splice or modify antibody genes at the molecular level, or artificially synthesize and introduce them into recipient cells for expression. The resulting new antibodies, also known as the third generation antibody.
Successfully studied genetically engineered antibodies include: chimeric antibodies, reconstituted antibodies, and small molecule antibodies.
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