PRODUCTION OF MONOCLONAL ANTIBODIES

Monoclonal antibodies are antibodies produced by single clone of cells or cell line and consists of identical antibody molecules.  Production of monoclonal antibodies is done using hybridoma technology. To understand hybridoma technology let us first understand what a HAT medium is.

HAT MEDIUM

HAT medium is one of the several important medium used for selection of hybrid cells. The

production of monoclonal antibodies

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medium is supplemented with hypoxanthine, aminopterin and thiamine. Hence, named HAT. Antimetabolite aminopterin blocks cellular synthesis of purines and pyramidines from sugars and aminoacids. However normal human and mouse cells can still multiply using hypoxanthine nad thiamadine present in the medium through salvage pathway, which helps in recycling of reduced from degradation of nucleic acids.
Hypoxanthine is converted to guanine by the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT) while thiamadine is phosphorylated by thiamadine kinase(TK). Both HGPRT and TK are enzymes involved in salvage pathway
On a HAT medium only those cell that are HGPRT+ and TK+ will grow, while those deficient in these enzyme can’t survive as they cannot produce purines and pyramines denovo (aminopterin blocks) or by salvage pathway.
HAT medium is used for the selection of hybrid cells in hybridoma technology.

production of monoclonal antibodies
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HYBRIDOMA TECHNOLOGY
This technique includes the production of hybrid used by fusion of B lymphocytes with a myeloma cell. The hybrid cells thus produced have the ability to produce antibodies (due to B lymphocyte genome) and grow infinitely invitro( due to tumor cell genotype).
Hybridoma cells are either cultured invitro or passaged through mouse peritoneal cavity to obtain monoclonal antibodies. This is known as hybridoma technology.
This technique was developed by G Kohler and C Mylstein in 1975 for which they won the Nobel prize in medicine.
The myeloma cells are selected for 2 features
1. These cells will not produce antibodies themselves.
2. They must have genetic markers.
When these cells are fused the resultant population has hybrid cells, myeloma cells and b lymphocyte cells. The cells are then cultured in  HAT medium that contains the drug aminopterin. The HGPRT– cells are unable to survive and divide in the HAT medium . Similarly, B lymphocyte do not grow for a longer period and eventually die. . Only hybridoma cells that are HGPRT+ and TK+ proliferate in the HAT medium.
The next step involves the identification and isolation of hybridoma cells. The cells are.suspended in microwells and then allowed to grow. The hybridoma cells grow and secrete antibodies against a specific antigen. The supernatant from each microwell is assayed for the presence of antibodies specific to antigen being used which result in either precipitation or agglutination reaction.
ELISA is the most efficient , rapid and sensitive assay used to analyse the reaction.

Monoclonal antibodies (from hybridoma cells) are mass cultured invivo in the mouse peritoneal cavity and invitro in large scale culture vessels.

INVIVO CULTURE OF MONOCLONAL ANTIBODIES
The hybridoma cells are transplanted to peritoneal cavity of appropriate strains of mice and subsequently ascytic fluid from animal is harvested. This method yields 50-100 fold higher antibody yield than invitro culture of hybridoma cells. This technique however has certain disadvantages,
1. The antibody preparation are of lower purity.
2. The procedure is very tedious.
3. Pathogen free animals are needed.

INVITRO CULTURE OF MONOCLONAL ANTIBODIES
Hybridoma cell can be cultured at large scale using stirrer bioreactor, airlift fermentor and in vessel based on immobilized cells. The culture system using immobilized cells enable the production at higher densities.

Monoclonal antibodies serve diagnostic and theraputic purposes. They are also used in immunopurification experiments. Hybridoma technology is one the bests techniques to be invented and has a wide range of applications in the science field.

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