Monoclonal antibody production involves the process of immortalizing B cells to create a stable cell line capable of generating identical antibodies. When B lymphocytes are exposed to antigens, they can differentiate and proliferate into plasma cells that secrete specific antibodies against those antigens. However, this ability is limited in terms of both duration and quantity. As a result, these cells cannot sustain long-term antibody production. To overcome this limitation, B cells are fused with non-secretory myeloma cells, forming hybridoma cells. These hybridomas combine the unlimited growth potential of tumor cells with the ability of B cells to produce specific antibodies. Once cloned, these hybridoma cells can be cultured or injected into animals to generate large quantities of high-titer, single-specificity monoclonal antibodies. This method is widely known as monoclonal antibody technology.
In our department, we have successfully prepared monoclonal antibodies for various antigen types. The primary focus has been on small molecule veterinary drug residues, but we also work with macromolecular immunoglobulins, viruses, vaccines, spores, peptides, and other targets.
**Preparation Process**
1) **Immune Spleen Cell Preparation**: Mice, typically Balb/c strain, are used as the source of B cells. The immunization method varies depending on the antigen type. Common approaches include subcutaneous or intraperitoneal injections, and in some cases, direct spleen injection may be employed.
2) **Myeloma Cell Culture and Screening**: Before fusion, myeloma cells are screened using HAT medium containing 8-azaguanine (8-AG) to eliminate mutant cells. Only those that can restore hypoxanthine-guanine phosphoribosyltransferase (HGPRT) activity survive. Myeloma cells are maintained in 10% fetal bovine serum culture medium and refreshed every 24 hours to ensure they remain in the logarithmic growth phase.
3) **Cell Fusion**: This step is critical and highly sensitive. Technical errors, such as poor immune response from B cells, can lead to failure. Contamination is another major cause of failure, so strict aseptic techniques and a clean environment are essential. The condition of the myeloma and spleen cells, their ratio, and the quality of the fusion agent all play crucial roles in successful fusion.
4) **Screening of Positive Clones**: Early screening is important, typically starting around 10 days post-fusion. False positives can occur if testing is done too soon. The chosen detection method should be accurate, sensitive, and efficient. ELISA is commonly used for its simplicity, while RIA offers higher accuracy. Multiple rounds of screening are necessary to confirm positive clones, which are then expanded once confirmed.
5) **Cloning and Stabilization**: Cloning is performed early and repeated to ensure a stable hybridoma cell line. Initially, hybridoma cells may be unstable and prone to chromosome loss. Repeated cloning helps establish a consistent and reliable cell population.
6) **Ascites Production and Antibody Purification**: Monoclonal antibodies are often harvested by inducing ascites in mice. Alternatively, large-scale in vitro cultures can be used depending on requirements. Initial purification is usually done using ammonium sulfate precipitation or zinc sulfate methods to concentrate and purify the antibodies.
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