Monoclonal Antibodies Production Steps by Hybridoma Technology

Antibodies are produced against antigen. whenever an antigen enters our body our immune system produces antibodies against that antigen. In normal condition as antigen enters it triggers or stimulates our immune system and our immune system produce antibodies.

  These antibodies are of different specificities and they recognise different epitops on the same antigen. The antibodies derived from the multiple cell are known as polyclonal antibodies. Polyclonal antibodies represent a collection of antibodies from different B-cells, that  recognise multiple epitops on the same antigen.

Monoclonal antibodies

preparations contain only one type of antibody derived from a single cloned B-cell. So these antibodies will recognise and bind only one particular epitop on an antigen. Monoclonal antibodies with same specificity are identical antibodies.

  Cesar Milsten and Grorges J.F. Kohlar described the first technique developed for stable Monoclonal antibodies production in 1975. This technique is known as Hybridoma Technology. They were jointly awarded Nobal Prise in physiology and medicine in 1984.

Hybridoma technology is used for the production of large number of monoclonal antibodies against a particular antigen and these monoclonal antibodies are used for the different purpose. i.e. in the treatment of cancer, used as anti-venoms.

Production of Monoclonal Antibodies by Hybridoma Technology

Production of Monoclonal Antibodies by Hybridoma Technology

1). Animal Immunization :

   First step in production of Monoclonal antibodies is the immunization of an animal, usually the animal is used as the mouse. Mouse is immunized with the antigen against which we need antibodies.

   Mouse is injected with the antigen (i.e. antigen which has three epitops) several time, as a result mouse B-lymphocytes are stimulated against the epitops or antigenic determinants of the injected antigen.

  After several weeks when these B-lymphocytes reach to an optimal amount the mouse is sacrificed, spleen of mouse is removed aseptically. Spleen is secondary lymphoid organ and we can easily harvest activated B-lymphocytes from spleen.

  Then we have activated B-cells which are capable of producing antibodies of against the specific epitops present on the antigen. Activated B-cells produce the antibodies of interest but can not replicate in culture.

2). Cell Fusion :

   In this step activated B-lymphocytes are fused with myeloma cells. The rational behind fusing these two cell type togather is that spleen cells produce the antibodies of interest but can not replicate in culture. Thus it is difficult to harvest antibodies from them. Myeloma cells on the other hand do not produce antibodies but to replicate in culture quite easily.

  Hybridomas take advantages of the properties of both cell types to mass produce is antibodies of interest.

As a result of cell fusion, we will have five type of cells:
-  Unfused B-cells,
-  Fused B-cells,
-  Unfused Myeloma cells,
-  Fused Myeloma cells and
-  Hybrid cells formed by fussion of an activated B-cell and in myeloma cell.
These hybrid cells are also known as Hybridomas

In our illustration we have five types of activated B-cells each specific to one of the three epitops on the antigen. So hybrid cells will also be of three types.

3). Selection of Hybridomas 

    Next aim is to select these hybridomas from this mixture of cells. Selection of hybridomas from the mixture can be accomplished through the use of media containing Hipoxanthine, Aminopterin and Thymidine (HAT).

  In order to understand the rational behind this approch it is important to know that mammalian cells can synthesize nucleotide using two different pathways that are -
  • De novo pathway and
  • Salvage pathway.

  Under normal conditions mammalian cells will use the de novo pathway to replicate. but de novo pathway is blocked by Aminopterin, when the de novo pathway is blocked cells will then utilize the salvage pathway as an alternative means to replicate. but this will only happen if Hypoxanthanine and Thymidine are present. 

  The kye to this approch is to use myeloma cells that are deficient in an enzyme called HGPRTase (Hypoxanthine-guanine phosphoribosyl transferase) which is required for the salvage pathway in this scenario unfused myeloma cells that are deficient in an enzyme called HGPRTase.

Myeloma cells are unable to replicate because the de novo pathway is blocked by Aminopterin and the salvage pathway is blocked by deficiency and HGPRT.

  Fused and unfused B-cell died within few days because of their short lifespan, They are not able to devide indefinitely in cell culture. Fused and unfused myeloma cells also die. This is also because of de novo pathway is blocked by Aminopterin and the salvage pathway is blocked by deficiency and HGPRT.

  Hybrid cells almost survived in HAT medium these hybrid cells are able to synthesise nucleotide by the salvage pathway. The functional HGPRT enzyme is contributed by the activated B-cell partner and these cells are able to divide indefinitely.

4). Isolation of Hybridomas :

    Our aim is to select legate single antibody producing hybrid cell. we need to isolate these hybridomas and grow them individually. Therefore next step is the isolation of hybridomas of single specificity, this is done by a method known as limiting dilution.

  In this method the cells which are hybridomas are distributed in multi well culture plates at very density. This is done such that on an everage each will contain a single cell.

5). screening :  

  In this step these hybridomas are screened for the secretion of the antibody of desired specificity. This screening  is done mostly by two technique namely ELISA and RIA. Once the hybridoma cells producing the desired antibody are identified they are isolated and cloned.

6). Cloning and Propagation :

   In this step we have seperated clones of activated B-cells each producing antibodies of a single specificity in each case. The antibodies produced are known as monoclonal antibodies.

7). Characterization and storage of Monoclonal antibodies :

  This is the final step in the step these hybridomas and monoclonal antibodies are characterized and stored. Mostly they are stored in liquid nitrogen. Now these monoclonal antibodies  are ready to treating and diagnosing disease.