Monoclonal antibody preparation process and principle

Monoclonal antibodies (MAbs) are antibodies produced by needle-specific antigenic determinants. The monoclonal technique, also known as hybridoma technology, originated in 1975 and was founded by G.KÖhler and Milstein. The main principle is to use antibody-producing B cells to hybridize with tumor cells to form hybridoma cells to produce antibodies. The monoclonal antibody preparation process has the following steps, which are briefly described below.

1. Immunization of animals Immunization Animals are processes in which mice are immunized with the antigen of interest to produce sensitized B lymphocytes in mice. Female Balb/c mice, 6-8 weeks old, are generally selected for immunization according to a pre-established immunization protocol. The antigen enters the peripheral immune organs through the blood circulation or lymph circulation, stimulates the corresponding B lymphocyte clones, activates, proliferates, and differentiates into sensitized B lymphocytes.

2. Cell fusion The mice were sacrificed by eyeball ablation and bloodletting. The spleen was aseptically removed and squeezed in a dish to prepare a spleen cell suspension. The prepared syngeneic myeloma cells are mixed with mouse spleen cells in a certain ratio, and a fusogenic agent polyethylene glycol is added. Under the action of polyethylene glycol, various lymphocytes can be fused with myeloma cells to form hybridoma cells.

3. Selective culture The purpose of selective culture is to screen for hybridoma cells, generally using HAT selective medium. In HAT medium, unfused myeloma cells are unable to synthesize DNA by the salvage pathway and die due to the lack of hypoxanthine-guanine-phosphoribosyltransferase. Although unfused lymphocytes have hypoxanthine-guanine-phosphoribosyltransferase, they cannot survive long-term survival in vitro and gradually die. Only the fused hybridoma cells can survive and proliferate in HAT medium by obtaining hypoxanthine guanine phosphoribosyltransferase from spleen cells and having the property that myeloma cells can proliferate indefinitely.

4. Screening and cloning of hybridoma-positive clones Hybridoma cells grown in HAT medium, only a few cells secreting a predetermined specific monoclonal antibody, must be screened and cloned. Clonal culture of hybridoma cells is usually carried out by limiting dilution. Using sensitive, rapid, and specific immunological methods, positive hybridoma cells that produce the desired monoclonal antibody are screened and cloned and amplified. After comprehensively identifying the immunoglobulin type, subclass, specificity, affinity, epitope and molecular weight of the secreted monoclonal antibody, it is frozen in time.

5. Large-scale preparation of monoclonal antibodies The large-scale preparation of monoclonal antibodies is mainly carried out by in vivo inducing methods and in vitro culture methods.

(1) In vivo induction method Balb/c mice were firstly pretreated by intraperitoneal injection of 0.5 ml of liquid paraffin or pristane. After 1-2 weeks, the hybridoma cells were inoculated intraperitoneally. Hybridoma cells proliferate in the peritoneal cavity of mice and produce and secrete monoclonal antibodies. About 1-2 weeks, the mice were seen to have enlarged abdomen. A large amount of monoclonal antibody can be obtained by taking ascites with a syringe.

(2) In vitro culture method Hybridoma cells are cultured in a culture flask. During the culture process, the hybridoma cells produce and secrete monoclonal antibodies, collect the culture supernatant, and centrifuge to remove the cells and fragments thereof to obtain the desired monoclonal antibodies. However, this method produces a limited amount of antibody. In recent years, various new culture techniques and devices have emerged, greatly increasing the production of antibodies. After hybridoma cells are fused, they must be screened before use. Hybridoma cells are divided into two groups: one, screening for hybridoma cells; second, selecting hybridoma cells that produce specific antibodies in the primary hybridoma cells, the methods and principles of the two screening methods are different.

The above five steps provide a brief overview of the monoclonal antibody preparation process. The following describes the experimental principle of monoclonal antibodies. To prepare a monoclonal antibody, it is necessary to obtain a monoclonal B lymphocyte capable of synthesizing a specific antibody, but the B lymphocyte cannot be grown in vitro. The experiment found that myeloma cells can grow and multiply in vitro, and the cell hybridization technique is used to combine the myeloma cells with the immune lymphocytes to obtain hybrid myeloma cells. This hybrid cell inherits the characteristics of two parental cells. It has the characteristics of specific antibodies for the synthesis of B lymphocytes, and also has the characteristics that myeloma cells can proliferate in vitro, and the cells derived from single fusion cells are cultured and propagated. Groups that produce specific monoclonal antibodies against an antigenic determinant.

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