In this post, we discuss about how the monoclonal antibody producing hybridoma cells are selected using the HAT media.

Antibodies (also known as Immunoglobulins) are large proteins produced by immune cells, primarily plasma cells. They can bind specifically to the antigens and help neutralise them. Structurally they are made up of two light and two heavy chains held together by disulphide bonds.

Monoclonal antibiotic production
Fig 1: Structure of an Antibody.

An antibody is specific for an antigen, and this specificity is exploited in various biomedical and diagnostic techniques like ELISA, flow cytometry, RIA, Immunochemistry, etc. To meet the demand, there is a need to produce antibodies on large scale. The polyclonal antibodies can be obtained from the sera of animals which are immunised against the required antigen.

The monoclonal antibodies, which are homogenous or identical synthesized from a single B-cell clone. They recognize same epitopes, or binding sites, on the antigen. The monoclonal antibodies can be generated by a technique pioneered by Georges Kohler and Cesar Milstein in 1975.

In this technique a normal activated Antibodies producing B cells were isolated. These activated B cells are sourced from the spleen of the animal (usually a mouse) immunised with the antigen of the interest.

The primed B cells are fused with myeloma cells. Myeloma is the cancer of the bone marrow. The myeloma cells chosen for this experiment have some important features:

1. These myeloma cells are HGPRT negative, that is they cannot synthesise an enzyme called Hypoxanthine Guanine phosphoribosyltransferase (HGPRT) (explained later).

2. These myeloma cells have also lost their ability to synthesize antibodies.

3. As the myeloma cells are cancer cells they are capable of infinite growth or are ‘immortal‘.

The two cells, that is, the activated B cells and myeloma cells are fused using polyethylene glycol (PEG). There are five different possible fusion products;

  1. Unfused B cells
  2. Unfused myeloma cells
  3. B- B homokaryon
  4. Myeloma- myeloma homokaryon
  5. B cell- myeloma hybrid or Hybridoma

To select the hybridoma (heterokaryon) out of the unfused cells and the homokaryons a specifically designed HAT medium is used. The HAT medium contains:

  1. Hypoxanthine
  2. Aminopterin
  3. Thymidine.

Aminopterin is a drug which is inhibitor of de novo purine synthesis, hence kills cells. When the de novo pathway is hindered, the cell uses an alternative pathway called Salvage pathway (salvage means rescue) for nucleotide synthesis. Salvage pathway requires Hypoxanthine and thymidine as precursors.

(Just for info: Read more about Nucleotide metabolism)

If aminopterin is present in the media the cell uses salvage pathway for nucleotide synthesis using hypoxanthine and thymidine. The hypoxanthine is converted to purines requiring enzyme HGPRT and thymidine is converted to TMP using enzyme thymidine kinase.

Principle HAT media for monoclonal antibody

Fig 2: Synthesis of nucleotides ( Principle of HAT medium)

The fused and unfused myeloma cells (in blue, Fig 3) lack HGPRT and hence cannot utilise Hypoxanthine in the media to synthesize purines and cannot survive on HAT media.

The fused and the unfused B cells (red) will die off anyhow, as they have finite life.

Monoclonal antibody
Fig 3: Production and selection of Hybridoma.

Only the hybridoma will survive on the HAT medium as

  • it has functional HGPRT from the B cell partner
  • divide infinitely, property from myeloma partner.
  • and it will produce antibodies as well.

Such a beautifully designed experiment!!

The hybridomas are screened for the secretion of the antibodies against the particular Antigen. The screened hybridoma are then cloned and propagated.

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Kohler G. and Milstein C (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature. 256, 495–497.