Hybridomas are tools for antibody discovery and production. They allow the capture and immortalization of highly productive plasma cells (effector B cells) able to secrete large quantities of antibodies. However, the production of monoclonal antibodies in hybridomas needs to be tailored for each specific clone. In this article, we summarize the essential steps of antibody production in hybridomas as well as how this ability can be used for efficient in vitro production. Check our frequently asked questions (FAQs) page about hybridomas for a complete overview of all steps of this robust process for antibody generation.
Antibodies are the key players in the humoral immune response. Their production in naïve B cells (or B lymphocytes) is triggered by the presence of foreign molecules (antigens). The activation of these naïve cells typically proceeds via:
Ideally, hybridomas should be produced from mature B cells (plasma cells) activated in a T cell-dependent way. This response takes longer to develop and it is often dictated by the dosage and frequency of the immunizations, as well as, by the quality and stability of the immunogen.
In a nutshell, the development of a strong immune response is paramount for the production of highly productive hybridomas. But this process requires the linked recognition of the same antigen particle by both B and T cells; the former is the precursor of antibody-secreting plasma cells while the latter catalyzes the process of B cell activation.
The frequent exposure to an antigen allows the host to produce a strong and highly specific response and to develop immunological memory. This process starts when professional antigen-presenting cells (APC) capture and degrade the injected antigens into epitope-containing peptides. These epitopes subsequently complex with class II MHC molecules (major histocompatibility complex) and become exposed on the surface of professional APC (typically dendritic cells).
These cells then migrate to the lymph nodes through the vast network of lymph vessels. At each node, the APCs carrying the antigen-MHC complexes are free to interact with naïve T cells possessing compatible T cell receptors (TCRs). Subsequently, this interaction activates the naïve T cells which, in turn, differentiate into armed effector T cells.
This process of differentiation generates three distinct classes of effector T cells, the most important of which is the CD4 TH2 class that are ultimately responsible for activating B cells. But not all B cells can be activated through this process, only the ones that have previously internalized a compatible antigen through their membrane Ig, processed it into the corresponding peptide fragments, complex them with class II MHC, and carry the complex exposed on their surfaces.
Once the TH2 cells are further activated by the linked recognition of an antigen, the production and secretion of cytokines take place. In turn, these serve as chemical signals that trigger antibody affinity maturation and production in plasma cells or generate immunological memory in memory B cells.
At this point, the plasma cells lose their membrane receptors and start secreting pentameric IgM molecules, providing the first line of humoral defense against pathogens. Subsequently, further stimulation by specialized T cells leads to class or isotype switching allowing the production of different antibody classes including IgG, IgA, or IgE.
The fully mature plasma cells that actively secrete large quantities of antibodies (preferably from the IgG isotype) are located in secondary lymphoid organs such as the spleen and lymph nodes. These organs served as the preferred sources of antibody-secreting for hybridoma production.
After collection, the antibody-producing cells are fused with compatible myeloma partners deficient in HGPRT, a vital enzyme in the de novo nucleotide synthesis pathway. The process of cell fusion is often mediated by chemical (i.e. PEG) or electrical signals and the fused cell lines are recovered by selection in HAT medium (Hypoxanthine-Aminopterin-Thymidine). The last step of the process comprises screening for antibody activity and storage or reculturing of hybridomas for further studies.
Antibody production in hybridomas serves as a quick way of producing small quantities for additional studies or direct use in diagnostic applications (e.g. clinical flow cytometry, Western Blot, ELISA, etc.).
Producing large quantities of monoclonal antibodies can be achieved in several ways and it depends mostly on the nature of the clone and the structure of the antibody. In fact, most protocols need to be adjusted and optimized for each particular clone, making native production challenging and difficult to scale. The most conventional protocol requires a slow adaptation of the hybridoma cell line to the culture medium containing FCS (fetal calf serum), prolonging the viability of the hybrid cell lines in standard laboratory conditions.
FCS contains trace amounts of antibodies, in the way, two solutions are available to recover the antibodies of interest from hybridoma culture supernatants:
After elimination of the serum from the hybridoma growth medium, monoclonal antibodies can be easily recovered from culture supernatants by straightforward protocols of purification using resins with immobilized protein A or protein G.
Interestingly, some hybridoma clones are unable to grow in serum-free medium. In these cases, two solutions are also possible:
Hybridomas are important tools in antibody discovery and production. They allow capturing high-affinity antibodies produced in vivo in response to foreign molecules. Hybridomas additionally serve for the native production of small quantities of antibodies for further characterization studies or direct use in some diagnostic applications.
For many years, hybridomas have been prized for their versatility. They can be easily stored for long periods of time and also be used for the quick production of small quantities of monoclonal antibodies. However, some hybridomas can only subsist on the complex nutrients found in sera of animal origin making antibody purification extremely challenging.
Recombinant expression arose as a suitable alternative to hybridoma cell culture production and it has been quickly gaining ground over this method for the production of monoclonal antibodies especially for diagnostic applications.
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