32.4:
Hybridoma Technology
Hybridoma technology is used to mass-produce monoclonal antibodies that can only bind to a single epitope — part of an antigen that elicits an immune response.
The process starts with injecting a mouse with a target antigen and then harvesting its spleen after a few weeks to isolate antibody-producing B cells. These cells have a limited life span and cannot be cultured in a lab.
Next, the B cells are fused with myeloma cells — immortal and cancerous white blood cells, using electric pulses or polyethylene glycol. Thus, forming hybrid immortal, antibody-producing cells called hybridomas.
Hybridomas are further selected on hypoxanthine -aminopterin -thymidine, or HAT medium based on the presence of a functional hypoxanthine-guanine phosphoribosyltransferase or HGPRT enzyme.
In this medium, both the HGPRT negative myeloma cells and the HGPRT positive but short-lived B cells cannot survive for long. However, the immortal hybridoma cells with the HGPRT enzyme can proliferate.
After selection, hybridoma cells that produce monoclonal antibodies with a strong affinity for the target epitope are identified and used as starter cells for establishing antibody-producing cell lines.
32.4:
Hybridoma Technology
Hybridoma technology is used for the large-scale production of monoclonal antibodies. Monoclonal antibodies bind to only a single antigenic determinant or epitope. Such antibodies are used in research, diagnostics, and disease therapy. The hybridoma technology established in 1975 by Georges Köhler and Cesar Milstein was awarded the Nobel Prize in Medicine in 1984 for revolutionizing research and therapy.
Hybridoma Selection
Commonly used fusion techniques — electroporation, polyethylene glycol (PEG) mediated fusion, and the use of fusogenic viruses — generally produce hybridomas at a very low frequency. Fusion products thus have a high ratio of self-fused B-cells and myeloma cells, or unfused cells, with a low number of hybrid cells.
The hypoxanthine-aminopterin-thymidine (HAT) medium is used to select hybridomas by selectively allowing their growth. The aminopterin blocks the default nucleotide synthesis in cells. However, cells can utilize hypoxanthine and thymidine from the medium to synthesize nucleotides via the salvage pathway.
Myeloma cells deficient in the HGPRT enzyme cannot synthesize nucleotides via this pathway and thus, do not grow in HAT medium. Although B-cells produce functional HGPRT enzymes, they cannot divide indefinitely. Thus, only the hybrid cells with functional HGPT from the B-cells and immortality from the myeloma line can grow on the HAT selection medium. Such a culture of hybrid cells is called a hybridoma, which can be screened for monoclonal antibody production.
Hybridoma Screening
Hybridoma cultures from the HAT selection are plated onto a 96-well plate; each well containing only one cell. Using Enzyme-linked Immunosorbent Assay (ELISA), each cell is screened for the production of antibodies specific to the epitope of interest. Cells that test positive are then selected and grown in a larger culture vessel to establish hybridoma cell lines. These cell lines are a permanent source of unlimited monoclonal antibodies. Thus hybridoma technology is a convenient and cost-effective method for mass production of monoclonal antibodies.
Suggested Reading
- Mitra S, Tomar PC. Hybridoma technology; advancements, clinical significance, and future aspects. J Genet Eng Biotechnol. 2021 Oct 18;19(1):159.
- Zaroff S, Tan G. Hybridoma technology: the preferred method for monoclonal antibody generation for in vivo applications. BioTechniques. 2019 Sep;67(3):90–2.