The Detailed Preparation Method of Monoclonal Antibody

Since Kohler and Milstein established lymphocyte hybridoma technology, Hybridoma are typically made by fusing tumor cells with the B lymphocytes that has been immunized with specific antigen. The hybridoma will secrete antibody as B cell and it could survive for as long as tumor cells. Moreover, these cancerous B-cells all produce a single type of antibody, called monoclonal antibody (McAb). Compared with polyclonal antibody, monoclonal antibody has advantages in terms of high purity specific, reproducible, and could support large-quantity supply. The advantages of monoclonal antibody technology, not only in immunology has brought revolutions, but also gets a very wide range of applications in various fields of biomedical science, and promote the development in many fields.

Generally the protocol of preparation of monoclonal antibody is as below:

1. Immunizing mouse with antigens

2. Getting spleens from immunized mice

3. Adopt hybridization technique, myeloma cells and B lymphocytes were fused to get hybridoma. Hybridoma not only can secrete stable antibodies , but also can replicate as tumor cells, so that we could get a large number of homogeous antibodies;

4. Screening hybridoma cells to get hybridoma cells which could secrete higher titers antibody;

5. Identification and cloning of the screened hybridoma;

The monoclonal antibody from hybridoma was widely used in in various fields for its great merit and potential. The McAb can be prepared according to the demand, and  it will be helpful to establish stable cell line continuously supplied application. Compared to antiserum, McAb can reduce the cost and number of animals, moreover its high-purity, uniformity, and can be used as standard reagents.

After obtaining hybridoma cell lines, there are two ways to obtain monoclonal antibodies: intraperitoneal injection of hybridoma in mice and cell culture.

(1) Mice ascites

Hybridoma cells were inoculated into the abdominal cavity of mice, hybridoma proliferation in the abdominal cavity, obtain ascites purified antibody can be obtained. This method is easy to operate, and has high reproducibility, less cost, high reproductivity, but this way will hurt the animal, but also requires a lot of animals when need large-scale production of antibodies.

Cell culture

Hybridoma were cultured in the flasks in order to gather the antibody . This method is suitable for large-scale production of antibodies. However, antibodies obtained in this way the purity is not that high, but the antibody might be influenced by the cell culture supernatant containing solution, such as pH indicator, cell metabolism waste. For this sake, cell culture will gradually reducing the serum medium to serum-free medium.

In summary, high specific monoclonal antibody will have promising prospects in  biological research and medical direction.

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