Hybridoma technology is a common method to produce monoclonal antibodies (mAbs) and involves the fusion of antibody-producing B lymphocytes obtained from immunized mice with immortal myeloma cell lines. This fusion gives rise to hybridoma cell lines which are cultivated to produce mAbs targeted against a specific antigen. This process can be achieved in vivo or in vitro, depending on the specific requirements of the research or therapeutic application.
Over 90% of approved therapeutic antibodies in 2020 were produced using hybridoma technology, with a majority of them being either chimeric or humanized. They hold several advantages over other antibody generation methods like phage display and single B cell screening, such as preserving the genetic pairing of variable and constant regions, the ability to optimize the mAbs for therapeutic use in humans, as well as in vivo affinity maturation.
However, while popular, this technology can be inefficient, involving long screening procedures, suboptimal selection of cells secreting specific mAbs, and the challenge of validating mAbs quickly, in addition to requiring purified antigen targets. Despite this, hybridoma technology offers a cost-effective means of producing mAbs and are widely used in medicine, toxicology, animal biotechnology, pharmacology, and cell and molecular biology.
Key Steps in Hybridoma Technology
Immunization and B-Cell Harvesting: The process begins with the immunization of an animal, commonly a mouse, with the antigen of interest. This antigen can be a protein, virus, bacteria, or any other molecule that triggers an immune response. After several immunizations over a few weeks, the mouse produces B lymphocytes (B cells) that are capable of producing antibodies specific to the antigen. These B cells are isolated from the spleen or lymph nodes for the next step. At this point, the animal is sacrificed to extract the antibody-producing B cells.
Cell Fusion: The isolated B cells are fused with myeloma cells, which are immortal cancer cells that do not produce antibodies but can divide indefinitely. The fusion is achieved using a chemical agent like polyethylene glycol (PEG) or by applying an electric shock (electrofusion), facilitating the merging of the cell membranes. The resulting fused cells are hybridomas, combining the longevity of myeloma cells and the antibody-producing capability of B cells. This fusion process, however, is inefficient, with only about 1-2% of cells successfully fusing to form hybridomas.
Selection and Screening: Once cell fusion is completed, the hybridoma cells are cultured in a selective medium known as HAT medium (hypoxanthine-aminopterin-thymidine). This medium allows only the fused hybridoma cells to survive. Myeloma cells that did not fuse perish because they lack the necessary enzyme (HGPRT) to survive in HAT medium, and unfused B cells die out naturally due to their limited lifespan. This selective environment ensures that only the viable hybridoma cells remain.
Cloning and Expansion: After the hybridomas are isolated, they are screened for the production of antibodies specific to the target antigen. This screening is usually done through techniques such as enzyme-linked immunosorbent assay (ELISA), Western blot, or flow cytometry, which test for the desired antigen-antibody interaction. Once a hybridoma producing the desired monoclonal antibody is identified, it is cloned through limiting dilution to ensure that all cells in the culture are identical. These cloned hybridomas are then expanded in culture, allowing for large-scale production of monoclonal antibodies.
In Vivo or In Vitro Antibody Production: Monoclonal antibodies can be produced either in vivo or in vitro. In vivo, hybridomas are injected into the peritoneal cavity of mice, leading to the accumulation of ascites fluid, which is rich in antibodies. However, this method may introduce contaminants, such as mouse immunoglobulins. In vitro, hybridomas are cultured in bioreactors or flasks, which allows for a more controlled environment and yields highly pure monoclonal antibodies suitable for clinical applications.
Advantages of Hybridoma Technology
High Specificity and Sensitivity: The monoclonal antibodies produced by hybridomas are highly specific to a single epitope on an antigen. This specificity is crucial in applications like cancer therapies and diagnostic tests, where precise targeting is necessary to avoid off-target effects.
Reproducibility: Once a stable hybridoma cell line is established, it can continuously produce the same monoclonal antibody, ensuring consistent and reliable results over time.
Unlimited Production: Hybridoma cells are immortal, meaning they can divide indefinitely. This allows for the large-scale and long-term production of monoclonal antibodies, making the process scalable and efficient.
Versatility in Applications: Monoclonal antibodies produced through hybridoma technology have a wide range of applications, including:
Diagnostics: They are used in diagnostic tests to detect toxins, drugs, hormones, or pathogens such as bacteria and viruses.
Therapeutics: Monoclonal antibodies are critical in treating diseases like cancer, autoimmune disorders, and infectious diseases. Examples include rituximab for lymphoma and trastuzumab for breast cancer.
Research: Monoclonal antibodies are used in immunoassays like ELISA, flow cytometry, and Western blotting to detect and quantify specific proteins in research studies.
Challenges and Limitations of Hybridoma Technology
Immunogenicity: Monoclonal antibodies derived from mouse cells can trigger immune responses in humans. This problem has been addressed through the development of chimeric and humanized antibodies, which incorporate human antibody sequences to reduce immunogenicity.
Time-Consuming: The process of generating a stable hybridoma cell line can take several months, which limits its utility in urgent scenarios, such as rapid pandemic responses.
High Cost: The production of monoclonal antibodies using hybridoma technology is expensive, especially when scaling up for commercial therapeutic use.
Low Efficiency: Only a small percentage of B cells successfully fuse with myeloma cells to form viable hybridomas, making the process less efficient compared to modern recombinant technologies.
References:
Mitra, S., & Tomar, P. C. (2021). Hybridoma technology; advancements, clinical significance, and future aspects. Journal of Genetic Engineering & Biotechnology, 19. https://doi.org/10.1186/s43141-021-00264-6
Moraes, J. Z., Hamaguchi, B., Braggion, C., Speciale, E. R., Viana Cesar, F. B., Soares, S., Osaki, J. H., Pereira, T. M., & Aguiar, R. B. (2021). Hybridoma technology: Is it still useful? Current Research in Immunology, 2, 32-40. https://doi.org/10.1016/j.crimmu.2021.03.002
