Hybridomas are the result of a fusion between an antibody producing spleen cell and an immortal myeloma cell.
How Hybridomas are Created
Immunization and B Lymphocyte Isolation
Hybridoma production begins by immunizing laboratory animals, typically mice, with the antigen of interest. This antigen stimulates the mouse’s immune system to produce B lymphocytes that generate antibodies targeting the specific antigen. After a series of immunizations, the spleen—rich in activated B cells—is removed for isolation of the B lymphocytes that now carry genetic information to produce antigen-specific antibodies.
Fusion of B Cells with Myeloma Cells
Isolated B lymphocytes are then fused with immortalized myeloma cells, which are cancerous plasma cells with a high proliferation capacity. This fusion process uses polyethylene glycol (PEG) to promote the merging of cell membranes, creating hybrid cells or "hybridomas." The myeloma cells provide longevity, while the B lymphocytes contribute the antibody-producing capacity. The hybridoma cells that form are effectively immortal, allowing for indefinite production of a consistent, highly specific monoclonal antibody.
Selection and Screening
Hybridoma cells are cultured in a selective medium (HAT medium) to filter out unfused cells. Only hybridomas—those with both B cell and myeloma cell properties—survive, as they possess both the antibody-coding genes from B cells and the longevity from myeloma cells. Once selected, the hybridomas are screened to identify those producing the desired antibody, which is subsequently purified for applications in research and therapeutics.
Advantages of Hybridoma Technology
Hybridoma-derived antibodies are highly specific
Reproducible and scalable, ensuring consistency across large batches
Unlimited production of monoclonal antibodies
Useful for highly sensitive and specific assays
Purity of antigen or immunogen is not a prerequisite
Not labor-intensive as in vitro antibody generation techniques uses immune libraries
Once the hybridoma line is established, the cost per antibody unit decreases
Widely used in both diagnostic and therapeutics1
Challenges in Hybridoma Technology
Time-consuming, taking 6-9 months
Expensive and requires considerable effort in production
Unsuitable for producing antibodies against small peptides and fragment antigens
High contamination risks
Currently only developed for mice and rats, but researchers are working to develop antibodies of human origin
Low viable efficiency of cells is quite low
Potential cross-contamination or infection in humans
Fusion of human lymphocyte and mouse myeloma cells may result in the production of unstable fused cells1
Related: What is Hybridoma Technology?
The Future of Hybridoma Technology
Advances in genome-editing tools, like CRISPR, offer the potential to improve the precision of human antibody production, reducing the need for traditional animal-derived hybridomas and potentially more diverse and human-compatible mAb therapies. As hybridoma technology continues to evolve, we can expect to see further improvements in monoclonal antibody production efficiency, specificity, and safety—transforming hybridoma technology from a classic method to a modern powerhouse in therapeutic antibody production.
Mitra, S., & Tomar, P. C. (2021). Hybridoma technology; advancements, clinical significance, and future aspects. Journal of Genetic Engineering & Biotechnology, 19, 159. https://doi.org/10.1186/s43141-021-00264-6
Antibody specificity refers to an antibody's ability to selectively bind to a unique epitope on a target antigen while avoiding interactions with unrelated antigens. This property arises from the highly specialized antigen-binding site located in the variable region of the antibody, which determines its unique binding characteristics.
Antibody affinity refers to the strength of the binding interaction between a single antigen epitope and the paratope (binding site) of an antibody. This interaction is a fundamental measure of how well an antibody recognizes its specific antigen target.
Recombinant antibodies are produced using genetic engineering techniques, unlike traditional antibody production, where the immune system generates antibodies without direct control over their sequence. By introducing genes encoding antibody fragments into host cells, such as bacteria or mammalian cells, recombinant antibodies can be expressed, purified, and deployed for applications including research, diagnostics, and therapeutics.
Recombinant antibody expression is a biotechnological process that involves engineering and producing antibodies outside their natural context using recombinant DNA technology.
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