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Hybridoma Technology Resources

What is hybridoma technology?

Hybridoma technology is an approach to producing monoclonal antibodies (mAbs) for specific antigen of interest. This well-established method was used to generate more than 90% of approved antibodies by the FDA [1]. The technology has evolved from developing monoclonal antibodies against murine antigens to a vast range of antigens in different species like rats, mice, rabbits, goats, sheep, cows and humans [1].  

In 1975, hybridoma technology was developed by Georges Kohler and Cesar Milstein. Milstein wanted to find a way to solve a key issue that was limiting the field of antibody research, the ability to isolate and purify single antibodies of known specificity. Although there were several known methods for cloning B cells to produce mAbs, those procedures had disadvantages such as short cell lifespan and low antibody yield [2].

Fortunately for Kohler and Milstein, mouse myeloma cells became available for them to test Milstein’s fusion theory. Milstein’s idea consisted of combining an immortal myeloma cancer cell to a short-lived antibody producing spleen B cell. This would give the new hybrid cell the properties of expressing a large amount of a specific mAb from the B cell and the immortality of myeloma cell [1].

Hybridoma technology was quickly adopted and widely utilized in the field of research, diagnostics and therapeutics.  For this discovery, Georges Kohler and Cesar Milstein were awarded the Nobel Prize in 1984 for their great contribution to the science community.  

At Abterra Biosciences, we have developed a cost effective high-throughput workflow that can sequence up to 96 hybridomas. The results include the full sequence of the variable region, the dominant and minor sequences for each hybridoma. It is common that a hybridoma comprises more than one monoclonal antibody. Therefore, we have included in our analysis the assessment of clonality of your hybridomas. Read more about a recent assessment of 185 hybridomas in When monoclonal antibodies are not monospecific: Hybridomas frequently express additional functional variable regions.

 

 

 

Generation of Hybridomas 

Hybridoma generation can be a labor-intensive process. There are 6 stages of production for generating and identifying high-quality hybridoma clones [3]. 

1. Immunization of Animals (generally mice)

The schedule of injections and quantities of antigen injected are dependent on the investigator and subjected to IUPAC approval. Over the course of 2-3 weeks, the mice will be exposed to a specific antigen through injections. The injection frequency is dependent on the protocol of the investigator. During that time, their bodies will build an immune response by creating antibodies that bind to that antigen. A type of white blood cell that produces antibodies is called B cell [4]. 

2. Screening for Antibody Production

After a couple of weeks of immunization, blood samples are taken for the measurement of serum antibodies. Serum antibody titer is usually determined by running an enzyme-linked immunosorbent (ELISA) assay or flow cytometry. Once a sufficient antibody titer is reached, the spleen and other tissue such as lymph nodes get harvested for the B cells to get fused with immortal myeloma cells [4]. 

3. Preparation of Myeloma Cells for Fusion 

A week before the fusion, myeloma cells are cultured and grown in 8-azaguanine to prepare them for the selection medium post cell fusion. These cells are selected to ensure they are not producing antibodies themselves and that they lack the hypoxanthine-guanine phosphoribosyltransferase (HGPRT) gene which makes them sensitive to hypoxanthine-aminopterin-thymidine (HAT) medium. Only the hybrid cells would survive in HAT media due to the functional HGPRT gene coming from the B cell. Unfused B cells have a short life span and would die on their own [4].  

4. Fusion of B cells with Myeloma Cells to Create Hybridomas

There are different techniques for cell fusion. The two preferable ones are electrofusion and chemical fusion. Electrofusion applies an electric field to cells in suspension which causes them to align and fuse. Chemical fusion uses polyethylene glycol (PEG) to dehydrate the cells and fuses the cell membranes as well as the intracellular membranes. For this method, the B cells from the spleen would be spun together with the prepared myeloma cells in PEG [3]. Newly fused cells get incubated in HAT media for 10-14 days and then they go through a limiting dilution step into 96 well plates. Ideally, the dilution would leave each well with one hybridoma cell.

5. Screening for Desired Antibodies

For this step, the hybridomas from the plates go through a rapid primary screening process to select the ones that produce antibodies of desired specificity. ELISA is typically used to identify positive hybridomas before cloning them to produce many identical daughter clones [3].  

6. Expansion of Clones for Harvesting Monoclonal Antibodies 

After the selection of positive clones, these individual hybridomas are transferred to larger tissue culture flasks. Each flask that contains a specific hybridoma cell line would produce monoclonal antibodies that have their own specificity. The cells can be propagated for cryopreservation and the supernatant can be used for investigational assays [3]. 

References:

  1. Parray HA, Shukla S, Samal S, et al. Hybridoma technology a versatile method for isolation of monoclonal antibodies, its applicability across species, limitations, advancement and future perspectives. Int Immunopharmacol. 2020;85:106639. doi:10.1016/j.intimp.2020.106639. (PubMed)
  2. Leavy, O. The birth of monoclonal antibodies. Nat Immunol 17, S13 (2016). (Nature)
  3. Steele T. Life Begins at Forty Hybridomas: Aging Technology Holds Promise for Future Drug Discoveries. (GaBI Journal)
  4. National Research Council (US) Committee on Methods of Producing Monoclonal Antibodies. Monoclonal Antibody Production. Washington (DC): National Academies Press (US); 1999. 1, Generation of Hybridomas: Permanent Cell Lines Secreting Monoclonal Antibodies. (NCBI)

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Antibody Reference Guide (downloadable)

Hybridoma Resources

Hybridoma Technology

Hybridoma Sequencing

Sequencing Pitfalls

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