ANTIBODY GENERATION

ANTIBODY GENERATION

12.      ANTIBODY GENERATION

If we want to generate an antibody against a particular antigen then we need to immunize the experimental live model (animal) with that antigen one or more times. As a result, after some time antibody against that antigen gets generated by antigen-specific multiple B cells in that animal and through bleeding that animal, the antibody is purified from the serum of the animal’s blood.
This type of antibody is called polyclonal antibody and this antibody identifies different epitope on that antigen, in turn, cause cross-reactivity and this cross-reactivity problem are overcome by the production of monoclonal antibody (mAb) through the stimulation of single type of B cell. As a result, mAb identifies single epitope present on an antigen. 
Georges Kohler and Cesar Milstein in 1975, identified the method for the generation of mAb and this method is known as hybridoma technology. In this technique hybrid of myeloma cell (a cancerous plasma cell) and single B cell, in turn, produce a hybrid cell that has immortal property along with the ability to produce mAb life long.  
12.1.    Steps of hybridoma technology
Myeloma cells used in this process are grown such that they are lacking the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT). As a result, they are not able to carry out nucleotide synthesis by salvage pathway because HGPRT is an obligatory enzyme for the salvage pathway. But mAb producing B cell are taken from the mouse spleen having HGPRT enzyme. This mouse was previously immunized with the antigen against which we want to make mAb.

Three types of combination can form, (A) two B cell fuse with each other, the hybrid cell produced by this fusion is able to synthesize mAb but mortal, thus die after some generation. (B) two myeloma cells fuse with each other, the hybrid cell produced by this fusion is immortal but is not able to produce nucleotide not only by salvage pathway because of lack of HGPRT enzyme but also by de novo pathway due to the presence of aminopterin, thus cells die. (C) One myeloma cell fuse with one B cell, the hybrid cell produced by this method is immortal as it is able to produce mAb and synthesize nucleotide by salvage pathway.  
Aminopterin is an antibiotic which inhibits nucleotide synthesis through the de novo pathway. In the presence of this antibiotic, neither B cell nor myeloma cell can synthesize nucleotide by the de novo pathway. Aminopterin present in a medium called HAT is used as a selective medium in which only hybrid cell grows. The HAT medium also includes thymidine and hypoxanthine, both required for nucleotide synthesis. 
Thus this hybrid cell survives and the clone of this cell has obtained the form by culturing of those cell in microtiter wells, as each well contain only one hybridoma cell. After that screening of those cells is done to check the ability to produce mAb by ELISA or RIA and now the clones able to produce mAb are selected and grown either in-vitro through culturing in suitable medium or invivo through inject in those clone cell into peritoneal cavity of mouse which is histocompatible and allowed to produce mAb through multiplication. In final step isolation of mAb is done by affinity chromatography.


Next Previous