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

Assignment on
Production of Monoclonal Antibody Using Hybridoma Technology
PHRM 407
Pharmaceutical Biotechnology
Section:1
Course Instructor
Najneen Ahmed (NNA)
Senior Lecturer
Department of Pharmacy
East West University
Prepared by
Md. Ahnaf TahmidID: 2016-1-70-054
Date of Submission: 6th November, 2018
Table of Contents
Topic Page No
Introduction 3
Immune System (Antigens, Antibodies, Polyclonal Antibodies Monoclonal Antibodies) 3
Hybridoma Technology 4
Production of Monoclonal Antibodies 4-6
Conclusion 7
Bibliography 8

Introduction
Antibodies are soluble protein components of humoral immune system. They possess highly specific nature to defend against foreign invaders such as microorganism and body’s own cells. 5
Immune system
Host defense mechanism that recognizes harmful molecules and give resistance against them on further occurrence. There are two effector mechanisms mediate the immune response.

Cell-mediated immune response (CMIR):
It is mediated by T-cells (T-lymphocites)
Antigen specific immune response 5
Humoral immune response (HIR):
It is mediated by antibodies
B-cells (B-lymphocites) produces antibodies 5
Antigens
An antigen is a foreign substance that can initiate the host cell defence mechanism. They possess a particular ‘Determinant (Epitope)’ on their surface which is complementary in structure to the receptor of lymphocites. Binding of antigens to the lymphocites depend on the epitopes. An antigen can have multiple epitopes on their surface. 1
Antibodies
Antibodies are protective proteins produced from B-cells when immune response is triggered. Upon binding of an antigen to an ‘Antigen Receptor’ on B-cells surface, B-cells multiplied and matured into a group of identical cloned cells. These identical matured B-cells then produce millions of antibodies and secretes into blood and lymph. Antibodies then neutralize the invading antigens by different methods depending on immunogen nature. Antibodies are also called immunoglobulins. 2
Polyclonal Antibodies
An antigen contains multiple epitopes which ultimately causes production of various antibodies in host organism. This causes due to the formation of multiple clone B-cells. So, the antibodies possessing different specificity for different epitopes of a specific antigen are called polyclonal antibodies. 5
Monoclonal Antibodies
Antibodies showing affinity for a single epitope of a specific antigen which is derived from a single B-cell clone is called monoclonal antibody. They are monovalent in nature. 5
Hybridoma Technology
Monoclonal antibodies can be produced using specially engineered cells called ‘Hybridomas’. This technique is called Hybridoma Technology. It was first discovered in 1975 by two scientists. One from Germany named Georges Kohler and another from Argentina named Cesar Milstein. They jointly awarded the Nobel prize in 1984 in the field of physiology and medicine. 6
Production of Monoclonal Antibodies Using Hybridoma Technology
The steps required in hybridoma technology are described below:

A mouse is injected with a specific antigen several times.

When the B-lymphocites are optimum in host organism spleenocytes from the spleen are collected from the mouse. Spleen cells are collected because B-cells are abundant in spleenocytes.

After spleen cells collection, spleenocytes are subjected to enzymatic or mechanical disruption to collect B-cells.

Density gradient centrifugation is used to separate the activated B-cells (plasma cells) from the medium.

Finally, we have the B-cells that can produce antibodies upon recognizing specific epitopes present in antigens. 3 4

Activated B-cells have short life span in culture medium. To sustain B-cells, they are fused with mutated myeloma cells. Mutated myeloma cells cannot produce own antibodies against activated B-cells due to mutation and they are HGPRT-
Cell fusion is done in a medium of 50% polyethylene glycol (PEG) for sufficient time.

There will be five types of cells in the medium after fusion process is completed
Unfused B-cells
Fused B-cells
Unfused myeloma cells
Fused myeloma cells
Hybrid cells (Hybridomas)
Target cells are hybridoma cells. But hybridomas will be of different epitope specific of an antigen. Now the mainstay will be separating hybridomas of single specificity. 3 4

HAT selection. H=Hypoxanthine A=Aminopterin T=Thymidine. HAT medium is HGPRT+ (Hypoxanthine-guanine phosphoribosyl transferase enzyme).
Aminopterin drug is used in HAT medium which blocks the pathway for normal nucleotide synthesis but opens another pathway which is dependent on HGPRT enzyme.

Cells from the fusion step are placed in HAT medium.
1866900215201500Fused and unfused B-cells will die within few days because they do not possess the tumorigenic property.

Also, the myeloma cells both fused and unfused will die because they are HGPRT-
Only hybridoma cells will survive in HAT medium because they are HGPRT+ and also possess tumorigenic property. As a result, they will be able to synthesize nucleotides. 6
-212090411480 Fig. Steps of monoclonal antibody production.
Adapted from: https://biocyclopedia.com/index/genetics/genetic_engineering_and_biotechnology_hybridoma_and_monoclonal_antibodies/hybridoma_and_the_production_of_monoclonal_antibodies.php Accessed 6 Nov. 2018.

020000 Fig. Steps of monoclonal antibody production.
Adapted from: https://biocyclopedia.com/index/genetics/genetic_engineering_and_biotechnology_hybridoma_and_monoclonal_antibodies/hybridoma_and_the_production_of_monoclonal_antibodies.php Accessed 6 Nov. 2018.

The step starts with a technique called ‘Limiting Dilution’.

Hybridoma cells survived from the HAT medium are placed in multi-well culture plates at very low density (On an average, each plate will contain a single cell). The culture plates are made of plastics.

Only one culture in several hundred will produce the target antibody that is specific for a single epitope. 3 4

Two techniques can be used ELISA and RIA. The most common technique is ELISA.

In this technique, the antigen of interest is adsorbed to the bottom of multi-well culture plates. A subsequent antigen-antibody binding reaction will occur if antibody presents. Other unbound materials will be washed off and eliminated. 6

This step again starts with ‘Limiting Dilution’ technique. Here, the antibody producing hybridomas are allowed to regrow and run the screening multiple times to ensure that the clones produce antibodies are monoclonal.

After assurance, the clones are then transferred into new wells and produce enough antibodies to characterize.

Hybridomas can also be freeze for future use. 5

In the final step, monoclonal antibodies are characterized and stored.

Characterization is done by different methods such as spectrometric, chromatographic, electrophoretic etc. Characterization is needed to determine the purpose of monoclonal antibodies. Also, stability of monoclonal antibody is known from this step.

They are stored mostly in liquid nitrogen. 5 6
Conclusion
In the last 10-15 years a considerable amount of effort has been given to improve the yield in monoclonal antibody production. Techniques used in hybridoma technology are also being tried to optimize the whole process. Overall, monoclonal antibodies have now become a greater aspect in the field of diagnosis of diseases and in medicine. Till now, they are very expensive due to huge production cost. As a result, uses are limited. Lastly, the days are not very far away when monoclonal antibodies will be using hugely to maximize efficiency in the diagnosis and treatment processes. 6

Bibliography
Encyclopedia Britannica. (2018). Antigen | biochemistry. online Available at: https://www.britannica.com/science/antigen Accessed 6 Nov. 2018.

Encyclopedia Britannica. (2018). Antibody | biochemistry. online Available at: https://www.britannica.com/science/antibody Accessed 6 Nov. 2018.

Satyanarayana, U. (2014). Recombinant DNA and biotechnology. 1st ed. Elsevier Health Sciences APAC.

Funke, B. and Tortora, G. (2013). Study guide for microbiology: an introduction, 11th ed. by Gerard J. Tortora, Berdell R. Funke, Christine L. Case. 11th ed. Boston, Mass.: Pearson.

Kulkarni, G. (2002). Biotechnology and its applications in pharmacy. 46th ed. New Delhi: Jaypee Bros Medical Publishers, pp.46-52.

Biocyclopedia.com. (2018). Hybridoma and the production of monoclonal antibodies | Genetic Engineering and Biotechnology Hybridoma and Monoclonal Antibodies | Genetics, Biotechnology, Molecular Biology, Botany | Biocyclopedia.com. online Available at: https://biocyclopedia.com/index/genetics/genetic_engineering_and_biotechnology_hybridoma_and_monoclonal_antibodies/hybridoma_and_the_production_of_monoclonal_antibodies.php Accessed 6 Nov. 2018.