In 1975, Kohler and Milstein discovered that mouse myeloma cells were fused with mouse spleen cells immunized with sheep erythrocytes, and the resulting hybrid cells produced both antibody and immortalization, thus creating a monoclonal antibody hybridoma technology. This technological breakthrough has not only opened a new era for basic research in medicine and biology, but also provided new tools for the diagnosis, prevention and treatment of clinical diseases.

Preparation of monoclonal antibodies including animal immunization, cell fusion, selection of hybridomas, detection antibodies, cloning of hybridoma cells, cryopreservation, and mass production of monoclonal antibodies, after several months of a series of experimental procedures, following the preparation of the preparation The sequence of cloning antibodies is introduced one by one.

First, preparation before cell fusion

(i) Immunization programme

Choosing the right immunization protocol is critical to the success of cell fusion hybridization to obtain high quality McAbs. It is generally necessary to establish an immunization program for the first immunization two months before the fusion, and the immunization schedule should be based on the characteristics of the antigen.

1. Granular antigen is highly immune, and good immunity can be obtained without adjuvant. The following is an immunization program using cellular antigens as an example:

First immunization 1×107/0.5ml ip (intraperitoneal injection)

↓ 2~3 weeks later

Second immunization 1×107/0.5ml ip

↓ 3 weeks later

Strengthen immunity (three days before fusion) 1 × 107 / 0.5ml ip or iv (intravenous injection)

↓

Spleen fusion

2. Soluble antigens are weakly immunogenic, usually with adjuvants. Common adjuvants: Freund's complete adjuvant, Freund's incomplete adjuvant. The antigen and the adjuvant are required to be mixed together in the same volume, and ground into a water-in-oil nipple shape, and a drop is not easily diffused on the water surface to form a droplet indicating that the water-in-oil state has been reached. Commercialized Freund's complete adjuvant must be shaken before use to allow the precipitated mycobacteria to mix well.

Primary immunization Ag 1～50μg plus Freund's complete adjuvant subcutaneous injection

│ (generally 0.8 ~ 1ml 0.2ml / point)

After 3 weeks

The second dose is the same as above, plus the addition of incomplete adjuvant subcutaneous or ip

│ (ip dose should not exceed 0.5ml)

After 3 weeks

The third immunization dose is the same as above, without adjuvant, ip

│ (After 5 to 7 days, blood is taken to measure its potency and the immune effect is detected)

↓ 2~3 weeks later

Strengthen the immunization, the dose is 50 ~ 500μg, ip or iv

↓3 days later

Spleen fusion

At present, the immunization protocols for soluble antigens (especially some weak antigens) are also constantly updated, such as granulating or immobilizing soluble antigens, on the one hand, enhancing the immunogenicity of antigens, and on the other hand, reducing antigens. The amount of use. 2 to change the route of antigen injection, the basic immunization can be directly injected into the spleen. 3 using cytokines as an adjuvant to improve the immune response level of the body and promote the antigenic reactivity of immune cells.

(2) Feeding cells

In the process of preparing monoclonal antibodies, many stages need to be added with feeder cells. For example, in the process of screening, cloning and expanding culture of hybridoma cells, it is necessary to add feeder cells. Commonly used feeder cells are: mouse peritoneal macrophages (more commonly used), mouse spleen cells or mouse thymocytes, and some people use the mouse fibroblast cell line 3T3 after radiation irradiation as a feeder cell, which is convenient to use and irradiate. After that, it can be stored in a liquid nitrogen tank for a long time, and it will be used with recovery.