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Monoclonal antibody preparation technology

2020-07-08
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Service background

Monoclonal antibody (McAb) uses lymphocyte hybridoma technology to fuse immunized animal B lymphocytes and myeloma cells to form hybridoma cells. Through HAT screening, ELISA antibody detection, and subcloning, the antibody secreting function can be selected and can be propagated indefinitely. Hybridoma cells, producing monoclonal antibodies. The advantages of monoclonal antibodies are specificity, uniformity, high efficiency and unlimited supply. In basic research and clinical medicine in the fields of immunology, medicine, biology, etc., including the diagnosis, prevention and treatment of diseases (cancers, etc.), they all show great vitality.

Monoclonal antibody preparation

Monoclonal antibody preparation technology

The biggest advantage of mAb is its specificity, uniformity, high efficiency and unlimited supply. Basic research and clinical medicine in the fields of immunology, medicine, biology, etc., including the diagnosis, prevention and treatment of diseases (including cancer), have shown great vitality.

The monoclonal antibody preparation service provides customers with complete preparation options, not only for you to prepare monoclonal antibody strains, but also for you to provide a large number of high-purity antibodies for a long time, so that customers can choose the most suitable service production line according to their needs. This technical service mainly includes:

    -Immunization of Balb/c pure line mice;

    -Fusion of spleen cells and SP2/0 myeloma cells;

    –ELISA screening of antibody-producing cells;

    -Subcloning and expansion of antibody-producing cells;

    -Production of monoclonal antibodies by in vitro culture of designated cell clones or ascites tumor inoculation.

Monoclonal antibody technical process

1. Antigen epitope design: using unique analysis software, assisted by on-line analysis method, synthesizing the three-dimensional structure of protein, active site, hydrophilicity and other factors, multi-parameter predicting epitope, greatly improving the reliability of antigen epitope , Provides a strong guarantee for subsequent antibody preparation.

2. Peptide synthesis, including: standard peptide synthesis, peptide modification, treatment of different purity peptides, antigen peptides and their cross-linking with proteins, etc.

3. Preparation of recombinant protein antigen

(1) Construction of recombinant plasmid

(2) Plasmid transformation

(3) Induced expression

(4) Protein purification

(5) Protein identification

The resulting recombinant protein has high purity and activity, and can be used to immunize animals to obtain monoclonal antibodies.

4. Preparation of hybridoma

(1) Immune animals: The choice of immunized animals depends on the source of myeloma cell lines. The myeloma cells commonly used in China are all derived from Balb/c mice. The immunogen may be: soluble antigen, granular (cell) antigen.

(2) Culture of myeloma cells.

(3) Preparation of feeder cells.

(4) Cell fusion.

(5) HAT screening.

5. Screening for hybridoma cells secreting antibodies

Commonly used are: immunofluorescence, radioimmunoassay (RIA), histochemistry and enzyme-linked immunoassay (ELISA) method, etc., ELISA method is the most commonly used.

6. Clonal culture of hybridoma cells

Although the tested hybrid cell secretes antibodies, it may be a hybrid cell or an antibody secreted by the progeny of multiple hybrid cells, so it must be cloned and cultured to obtain a clone that is both cloned and secretes antibodies Hybridoma cell line.

7. Mass production of monoclonal antibodies

After the hybridoma cells are constructed, a large amount of antibodies need to be prepared for use. Inoculation of hybridoma cells into the abdominal cavity of mice, as the hybridoma grows, exudates a large amount of ascites. The ascites contains antibodies, which can reach the level of milligrams per milliliter, which is currently widely used.

8. Antibody purification

Generally speaking, monoclonal antibodies are already very pure and can be used in experimental research. However, due to their different sources, they may contain components of the culture medium or murine peritoneal fluid and other unrelated proteins that affect purity. Therefore, ammonium sulfate precipitation method is required. , Octanoic acid-amine sulfate method, DEAE-cellulose method or affinity chromatography and other methods for purification. The company mainly uses affinity chromatography to obtain high-purity monoclonal antibodies for customers.

9. Antibody identification

After the hybridoma cells are established, the physicochemical properties of the monoclonal antibodies secreted and the specificity of the antigens need to be identified. The following tasks are usually required: the determination of the IgG subclass of the antigen itself; the determination of the molecular weight of the anti-antigen ; Western blot identification; ELISA titer identification, etc.

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