Assignment on
Monoclonal Antibody Production Using Hybridoma Technology
PHRM 407
Pharmaceutical Biotechnology
Section: 1

Course Instructor
Najneen Ahmed (NNA)
Senior Lecturer
Department of Pharmacy
East West University

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Submitted by
Puspa Sornakar
ID: 2016-1-70-054

Date of Submission: 5th November, 2018
Introduction- Immunoglobulins are antigen binding proteins that binds with antigen. Whenever any antigen specially an extracellular antigen enters our body it stimulates a specific B cell that activate plasma cells to produce antibodies.

Video

Immune system- It is the defense mechanism of the body that fight against infectious organisms and other invaders. There are two effector mechanism that mediate the immune response.

Cell mediated immune response
Cell mediated
Controls intracellular pathogens
T-cell plays major role

Humoral immune response
Antibody mediated
Controls extracellular pathogen
B-cell plays major role
(Kulkarni, 2002)
Antigen- Antigen is a substance that trigger the immune system to induce immune response. It is also called immunogen as it instigates an immune response.
An epitope, also called antigenic determinant is the section of an antigen that has a definite 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 bind to immunogen and make them inactive by precipitating them. Antibodies are secreted by plasma cells and is an important part of the immune system.

Monoclonal antibody- Monoclonal antibodies are immunoglobulins that respond to only one specific epitope and derived from a single plasma cell. Monoclonal antibodies are obtained by using hybridoma technology. They can only identify 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.

Hybridoma technology
Monoclonal antibodies are produced effectively by using hybridoma technology.
Normally, plasma cells can be cultured on artificial media but they cannot subsist in artificial media and lifespan is too short. But in hybridoma technology the short lifespan of plasma cells is vanquished 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. These cells are then be cultured on artificial media.(Kulkarni, 2002)
Production of monoclonal antibody using hybridoma technology-
The steps required for the production of monoclonal antibodies-
Immunize the animal for antibody production
Cell fusion
Hybridoma cell selection
Screening
Cloning
Characterization and storage

1. Immunization of animal
First step of hybridoma technology is to immunize an animal with particular antigen. Usually mices are used.
A suitable adjuvant is selected and appropriate antigen is compounded with the adjuvant.
Then the antigen adjuvant mixture is injected subcutaneously at multiple sites.
Injections are given in a repetitive manner.
Thus, immunization of the mice will incite immune response.
B cell will be produced in response to the particular invader.
When optimal antibody concentration is achieved, the animal is immolated.
Its spleen is removed and lymphocytes of the spleen (spleenocyte) are collected by mechanical techniques.

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 cannot 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 cannot survive. But myeloma cells (both fused and unfused) have defective HGPRT. So, they cannot grow in the HAT media.
So now we will get only hybridomas from the medium.
These selected cells are then distributed into multi well culture plates and it is done in a way that each well contain a single cell.

4. Screening
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.

5. Cloning
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
Carbohydrate structure
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.
(Biology Discussion, 2018)

Conclusion
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.

Bibliography
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.

Kulkarni, G. (2002). Biotechnology and its applications in pharmacy. New Delhi: Jaypee Bros Medical Publishers, pp.46-52.
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.
Jana, S. (2018). HYBRIDOMA TECHNOLOGY ; ITS APPLICATION. online Academia.edu. Available at: http://www.academia.edu/6017218/HYBRIDOMA_TECHNOLOGY_and_ITS_APPLICATION Accessed 4 Nov. 2018.

YouTube. (2018). Hybridoma Technology: Animated explanation. online Available at: https://www.youtube.com/watch?v=cCeze8tA_XI;feature=share video Accessed 4 Nov. 2018.

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