Hybridoma technology

Hybridoma technology is a biotechnological method that involves the production of monoclonal antibodies (mAbs) for various applications in research, diagnostics, and therapy. This technology was developed in 1975 by César Milstein and Georges J. F. Köhler, an achievement for which they, along with Niels K. Jerne, were awarded the Nobel Prize in Physiology or Medicine in 1984. Hybridoma technology combines the specificity of antibodies produced by a single B-cell with the immortal nature of myeloma cells, resulting in a cell line that can produce large quantities of identical antibodies, known as monoclonal antibodies.

Overview

The process begins with the immunization of an animal, typically a mouse, with an antigen of interest. This stimulates the animal's immune system to produce B-cells that generate antibodies against the antigen. These B-cells are then harvested from the spleen of the immunized animal and fused with myeloma cells (cancerous B-cells that can proliferate indefinitely) in the presence of a fusion agent, usually polyethylene glycol (PEG). The resulting fused cells, known as hybridomas, are capable of both producing the desired antibody and replicating indefinitely.

Selection

Following fusion, the hybridoma cells are placed in a selective medium, such as HAT medium (hypoxanthine, aminopterin, and thymidine), which only allows the growth of hybridoma cells. Aminopterin blocks the nucleotide synthesis pathway, but hybridoma cells can bypass this blockage using the salvage pathway, thanks to the enzymes contributed by the myeloma cell parent. Unfused myeloma cells cannot survive because they lack the necessary enzymes, and unfused B-cells die because they do not have the capability to proliferate indefinitely.

Screening

The hybridoma cells that grow in the HAT medium are then screened to identify those producing the desired antibody. This is typically done using assays such as ELISA (enzyme-linked immunosorbent assay). Once identified, the positive clones are isolated and expanded. Each clone produces a unique monoclonal antibody, making it crucial to select the clone with the best characteristics for the intended application.

Production

For small-scale production, the selected hybridoma can be cultured in vitro in laboratory flasks. For large-scale production, the hybridomas can be grown in bioreactors or in the peritoneal cavity of mice, a method known as ascites production. The monoclonal antibodies are then harvested from the culture medium or ascites fluid, purified, and prepared for use.

Applications

Hybridoma technology has revolutionized the field of diagnostics and therapeutics. Monoclonal antibodies produced using this technology are used in a wide range of applications, including:

• Cancer therapy, where they can be designed to target specific cancer cells.
• Diagnostic tests, such as pregnancy tests and tests for infectious diseases like HIV and hepatitis.
• Research, to identify and isolate specific proteins, pathogens, or cells.

Challenges and Developments

Despite its significant contributions, hybridoma technology faces challenges such as the ethical concerns surrounding animal use and the production of antibodies that may not function well in human therapy due to differences between species. To address these issues, techniques such as recombinant antibody technology and phage display are being developed to produce human or humanized antibodies without the need for animal immunization.

Conclusion

Hybridoma technology remains a cornerstone in the production of monoclonal antibodies, with ongoing research aimed at improving the efficiency and ethical aspects of antibody production. Its contributions to medicine and research continue to be invaluable, offering precise tools for diagnosis, research, and treatment.