The first antibodies ever produced in vitro were murine monoclonal antibodies obtained using the hybridoma technology in 1975. Hybridomas have since become one of the most relevant technologies for antibody discovery. By leveraging the highly cost-effective and efficient process of affinity maturation, hybridoma-generated antibodies continue to be one of the best tools for a variety of applications including therapy and diagnostics. Check our frequently asked questions (FAQs) page about hybridomas for a complete overview of all steps of this robust process for antibody generation.

What is the origin of hybridoma cells?

Hybridomas are hybrid cell lines obtained by immortalizing antibody-secreting spleen cells. This delicate process renders the vulnerable antibody-secreting cells stable under standard laboratory conditions.

The hybridization process can be induced by chemicals or viruses and it allows capturing highly mature immune repertoires from a specific host. Most hybridomas are generated by immunizing mice or other rodents with proteins, immunogenic peptides, or DNA. However, recently, alternative host species such as rabbit or chicken have become available.

Nevertheless, mice continue to be the favored species for hybridoma generation since the protocol and cell lines have been fully optimized after decades of research.

How are hybridoma cells generated?

Hybridomas are generated by fusing spleen cells (plasma cells) from immunized hosts with a compatible myeloma cell line (malignant and immortal plasma cell line). The myeloma partner used in this process typically lacks hypoxanthine-guanine-phosphoribosyltransferase (HGPRT), an enzyme that allows cells to grow in HAT medium (Hypoxanthine-Aminopterin-Thymidine).

Cell fusion between myelomas and plasma cells is recurrently induced by electric pulses (electrofusion) or polyethylene glycol (PEG). Alternatively, the immortalization of plasma cells can be triggered by infection with the Epstein-Barr virus (EBV); nevertheless, this process is known to be less efficient than PEG-mediated cell fusion which remains the gold standard reagent for hybridoma generation.

How are hybridoma cells separated from unfused cells?

After cell fusion, three types of cells are generated: unfused myeloma cells (HGPRT deficient), unfused spleen cells (short lifespans), and fused hybridoma cells. The selection of hybridomas from this mixture depends on the modulation of the nucleotide synthesis process.

In mammalian cells, DNA synthesis is carried out via two distinct pathways:

  • De novo synthesis pathway – which depends on the activity of dihydrofolate reductase
  • Salvage pathway – which depends on the presence of exogenous hypoxanthine and thymidine and the enzymes HGPRT and thymidine kinase (TK)

In the HAT medium, aminopterin, a dihydrofolate reductase inhibitor, is used to inhibit de novo nucleotide synthesis pathway, while exogenous hypoxanthine and thymidine are supplied to ensure DNA synthesis can be carried out via the salvage pathway.

Hybridomas are thus selected by continuously feeding the mixture of the three different cell types with HAT medium (kept in 96-well microtiter plates in a carbon dioxide incubator at 37°C):

  1. Unfused plasma cells are easily eliminated in these conditions since they do not replicate in culture medium
  2. Unfused myelomas can be easily selected in HAT medium since the de novo pathway is blocked and the salvage pathway as well (absence of the essential HGPRT)
  3. Hybridomas are the only cell types able to survive in these conditions, they inherit the HGPRT from the plasma cells and the ability to grow indefinitely in standard laboratory conditions from the myeloma counterparts

How are hybridoma cell lines screened?

At this stage, even after the selection and elimination of the unfused cell lines, each well still contains a mixture of different hybridoma clones producing different antibodies. For this reason, screening becomes the logical next step in the hybridoma generation process.

Typically, antibody binding affinity is first determined using culture supernatants from each well which contains a mixture of antibodies with considerably different affinities towards the target (polyclonal stage). Screening of antibody affinity at this stage is performed using ELISA (enzyme-linked immunosorbent assay).

The wells exhibiting the highest affinity towards the intended target are then used as starting points for monoclone isolation (limiting dilution) and clonal expansion followed by validation in the target application.

Concluding remarks

Hybridomas are hybrid cell lines produced through the immortalization of antibody-secreting plasma cells either by a process of cell fusion with an adequate myeloma partner or infection with EBV. After immortalization, hybridomas must be separated from their unfused partners by a process of metabolic selection in HAT medium.

The initial screening of the polyclonal hybridoma populations is performed by ELISA, followed by monoclone isolation and clonal expansion to ensure only the best-performing cell lines are selected for further studies.

Ideally, these hybridoma-derived antibodies should be validated in the intended application (e.g. Western Blot, Flow Cytometry, Immunohisto– or Immunocytochemistry, etc.) and sequenced so they can be engineered for therapeutic applications (e.g. humanization) and/or adapted to recombinant expression in high-performing mammalian systems such as CHO (Chinese hamster ovary) or HEK (human embryonic kidney) cells.

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