Monoclonal antibody preparation service

Monoclonal antibodies (MAb) are antibodies produced by specific antigenic determinants, and monoclonal technology, also known as hybridoma technology, originated in 1975 by G. KOhler and Milstein. The main principle is to use B cells that produce antibodies to hybridize and fuse with tumor cells to form hybridoma cells, producing antibodies.

The main steps for preparing monoclonal antibodies using hybridoma technology include:

(1) Antigen preparation,

(2) Immune animals,

(3) Preparation of immune spleen cells and myeloma cells,

(4) Cell fusion;

(5) Selective cultivation of hybridoma cells,

(6) Screening of hybridoma cells;

(7) Cloning of hybridoma cells;

(8) Detection of monoclonal antibodies;

(9) Establishment of hybridoma cell lines secreting monoclonal antibodies:

(10) Large scale preparation of monoclonal antibodies.

The preparation process of monoclonal antibodies:

1. Immune animals are the process of immunizing mice with target antigens to produce sensitized B lymphocytes. Generally, 6-8 week old female Balb/c mice are selected and immunized according to a pre established immunization regimen. Antigens enter peripheral immune organs through blood circulation or lymphatic circulation, stimulating corresponding B lymphocyte clones to activate, proliferate, and differentiate into sensitized B lymphocytes.

2. Cell fusion was performed by enucleation and bloodletting of mice, followed by sterile extraction of the spleen. The spleen was then compressed and ground in a petri dish to prepare a spleen cell suspension. Mix the prepared homologous myeloma cells with mouse spleen cells in a certain proportion and add the fusion promoting agent polyethylene glycol. Under the action of polyethylene glycol, various lymphocytes can fuse with myeloma cells to form hybridoma cells.

3. The purpose of selective culture is to screen for fused hybridoma cells, usually using HAT selective culture medium. In HAT medium, unfused myeloma cells die due to a lack of hypoxanthine guanine phosphoribosyltransferase, which prevents them from utilizing the salvage pathway to synthesize DNA. Although non fused lymphocytes possess hypoxanthine guanine phosphoribosyltransferase, they cannot survive in vitro for a long time and gradually die. Only fused hybridoma cells can survive and proliferate in HAT medium due to obtaining hypoxanthine guanine phosphoribosyltransferase from spleen cells and possessing the characteristic of unlimited proliferation of myeloma cells.

4. Screening and cloning of hybridoma positive clones: Only a few hybridoma cells grown in HAT medium secrete specific monoclonal antibodies, therefore, screening and cloning are necessary. The limited dilution method is usually used for clonal culture of hybridoma cells. Using sensitive, rapid, and specific immunological methods, positive hybridoma cells that can produce the desired monoclonal antibodies are screened and cloned for amplification.

After comprehensive identification of the immunoglobulin type, subtypes, specificity, affinity, antigen recognition epitopes, and their molecules secreted by the monoclonal antibody, it is promptly frozen.

5The large-scale preparation of monoclonal antibodies relies heavily on animal in vivo induction and in vivo methods

External cultivation method.

(1) Balb/c mice were induced by intraperitoneal injection of 0 Pre treat with 5ml of liquid paraffin or decalin. 1-2 weeks later, hybridoma cells were inoculated intraperitoneally. Hybridoma cells proliferate in the abdominal cavity of mice and produce and secrete monoclonal antibodies. About 1-2 weeks, abdominal swelling can be seen in mice. Extracting ascites with a syringe can obtain a large amount of monoclonal antibodies.

(2) The in vitro culture method involves placing hybridoma cells in a culture bottle for cultivation. During the cultivation process, hybridoma cells produce and secrete monoclonal antibodies. Collect the culture supernatant, centrifuge to remove cells and their fragments, and obtain the desired monoclonal antibodies. But the amount of antibodies produced by this method is limited. In recent years, various new cultivation techniques and devices have emerged, greatly increasing the production of antibodies. After fusion of hybridoma cells, screening is necessary before use. Hybridoma cells are divided into two stages: first, selecting hybridoma cells; 2、 The methods and principles for selecting hybridoma cells that can produce specific antibodies from the initial selection of hybridoma cells are different.

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