Introduction- Immunoglobulins or antibodies are antigen binding proteins that binds with antigen to precipitate the antigen thus making it inactive. Whenever any antigen specially an extracellular antigen enters our body it stimulate a specific B cell that activate plasma cells to produce antibodies that can bind with that particular antigen.
Immune system- is the body’s defense mechanism against infectious organisms and other invaders. There are two effector mechanism that mediate the immune response.
Cell mediated immune response
Controls intracellular pathogens
T-cell plays major role
Humoral immune response
Controls extracellular pathogen
B-cell plays major role
Antigen- Antigen is a substance that stimulates the immune system specially production of antibody. It is also called immunogen because it provokes an immune response.
An epitope, also called antigenic determinant is the part of an antigen that has a particular molecular structure which is recognized by a specific immunoglobulin. Antigen may carry a single epitope or multiple epitopes.
Antibody- antibodies are the substances that are produced from B cells. These protein molecules binds to antigen and make the antigen inactive by precipitating that particular antigen. Antibodies are secreted by plasma cells and is an important part of the immune system.
Monoclonal antibody- Monoclonal antibodies are immunoglobulins that responds to only one specific epitope and derived from a single plasma cell. Monoclonal antibodies are obtained by using hybridoma technology. They can only recognize and bind with a single epitope whereas polyclonal antibodies can bind with different epitopes at the same time. Milstein produced the first monoclonal antibody in 1990.
Monoclonal antibodies are produced effectively by using hybridoma technology.
Normally, plasma cells can be cultured on artificial media but they can not survive in artificial media and lifespan is very short. But in hybridoma technology the short lifespan of plasma cells is overcome by fusing a normal active B cell and a myeloma cell. Thus the hybridoma cells have the immortal growth properties of the myeloma cell and antibody secreting properties of the active B cells. This cells are then be cultured on artificial media.
Production of monoclonal antibody using hybridoma technology-
The steps required for the production of monoclonal antibodies-
Immunize the animal for antibody production
Hybridoma cell selection
Characterization and storage
1.Immunization of animal
First step of hybridoma technology is to immunize an animal with particular antigen. Usually mouse are used.
A suitable adjuvant is selected and appropriate antigen is mixed with the adjuvant.
Then the antigen adjuvant mixture is injected subcutaneously at multiple sites.
Injections are given in a repetitive manner.
Thus immunization will provoke immune response.
B cell will produce in response to the particular antigen.
When optimal antibody concentration is achieved, the animal is sacrificed.
Its spleen is removed and lymphocytes of the spleen (spleenocyte) are collected.
2. Cell fusion
Spleenocytes are mixed with HGPRT defective myeloma cells.
In polyethylene glycol (PEG) the mixture is exposed for a short period of time.
Thus these two cells are fused together.
PEG is eliminated by washing and the cells are kept in fresh medium.
Now the medium may contain free plasma cells, fused plasma cells,free myeloma cell,fused myeloma cells and our type of interest hybridoma cells.
3.Hybridoma cell selection
For the selection, the cells will be transferred into HAT media. HAT stands for Hypoxanthine aminopterin thymidine.
For the cell division to take place in the artificial media, the cells must synthesize nucleotides for new DNA copy.
There are two pathways for the formation of nucleotides. Salvage pathway where new nucleotides are formed from degraded nucleotides and second one is de novo pathway where nucleotides are formed using small metabolites.
In HAT media, cells can not operate de novo pathway because aminopterin of the HAT media blocks a key enzyme that is dihydrofolate reductase. So cells undergo the salvage pathway which is HGPRT dependent. HGPRT enzyme uses hypoxanthine and thymidine as precursor to form new nucleotides.
Only the hybridoma cells can grow in HAT media. Because hybridoma cells are HGPRT positive. Plasma cells both fused and free also have HGPRT but due to their short lifespan they can not survive. But myeloma cells (both fused and unfused) have defective HGPRT. So they can not grow in the HAT media.
So now we will get only hybridomas from the medium.
This selected cells are then distributed into multi well culture plates and it is done in a way that each well contain a single cell.
The hybridoma cells are screened for the secretion of specific antibody.
There are two techniques for screening. One is ELISA and other one is RIA
In both assays the antigen is coated to plastic plates and the antibody binds to the specific antigen.
Thus the unbound antibody and other components of the medium is removed by washing.
In this way, the hybridoma cells that produce the specific antibody is identified.
The antibodies that are secreted from the hybridoma cells are called monoclonal antibodies.
Single hybrid cells producing antibody of single specificity are isolated and cloned.
Two methods are applied. They are limiting dilution method and soft agar method.
6. Characterization and storage
Biochemical and biophysical characterization of monoclonal antibody is done.
Structural characterization may include
Amino acid sequence
Sulphydryl groups etc
Physiological analysis may include
Molecular weight and size
Electrophoretic pattern etc
It is important to determine immunoglobulin sub-class.
MAbs should be characterized for their ability to withstand freezing, thawing etc.
The cells are stored in liquid nitrogen.
The hybridoma technology has open the way for the commercialization monoclonal antibodies. The one main problem with this technology is that it takes months to yield monoclonal antibodies. Research is going on to overcome this problem. Anyways, monoclonal antibody production can be considered as a milestone. They are being used widely in treatment of diseases like cancer, rheumatoid and also being used as a dignostic tool.
https://www.youtube.com/watch?v=8iyrbv1JauY;feature=share. (2018). video.
Reason, A. (2018). Structural Characterization of Monoclonal Antibodies. online Biopharminternational.com. Available at: http://www.biopharminternational.com/structural-characterization-monoclonal-antibodies Accessed 4 Nov. 2018.
Biology Discussion. (2018). Monoclonal Antibodies: Production, Advantages and Limitations. online Available at: http://www.biologydiscussion.com/antibodies/monoclonal-antibodies-production-advantages-and-limitations/10068 Accessed 4 Nov. 2018.