In 1975 Monoclonal antibody was first produced by Kohler and Milstein. When antibodies are made by identical immune cells which are clones of parent cell is called monoclonal antibody. For the production of monoclonal antibody hybridoma technology is used where B-cell is made immortal by fusing them with Myeloma cell. The fused cell is known as hybridoma.1
To produce monoclonal antibody, hybridoma technology is very important. B cell is involved here which can produce an antibody that have single specificity, Production of B cell or plasma cell can be impelled by the immunization of an animal with an antigen. The B cells produce specific antibodies for different epitopes of the antigen. But it is quite difficult to maintain these cells in culture medium, which prohibit production of MAbs. In this technology to make B cell immortal it is fused with myeloma cell which is an artificial medium. It can overcome the death of plasma cells in culture medium but not possible to control the cell become malignant, and the anitbody which produced are useless. 1
Hybrid cell formation by the fusion of normal and malignant cell can solve the problem. These hybrids were named as heterokaryons.1
Sendai virus was helped for this fusion but more efficient fusogen is PEG (Polyethylene Glycol). In the medium all other cells except the fused hybrid cell will die.1

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Hybridoma Production of Monoclonal Antibody: In monoclonal antibody production by hybridoma technology there are some following methods-

1. Immunization: Immunogen is injected into the laboratory mice. It is mixed with suitable adjuvant and injected intradermally or subcutaneously. This injection procedure should be in repeated manner. Blood sample is taken and assayed for the presence of the desired antibody. If the antibody is present and is in desired amount then the mice is sacrificed and it’s spleen is disintegrated into spleenocytes. 1

2. Cell Fusion: In this step the acquired spleenocytes are fused with plasmacytoma cells. This fusion takes place in a suitable medium like PEG (Polyethylene Glycol). The concentration of the medium must be high (about 50%). This mixture will later form the hybridoma.1

3. Selection: In the selection step the fused cells are incubated in HAT medium (Hypoxanthine-Aminopterin-Thymidine medium) for kind of 10 to 14 days. The viable hybridoma cells are formed in this medium. Afterwards they are transferred to a regular culture medium. The incubated medium is then diluted into multi-properly plates (96-well plastic culture plate) to such an volume that every well contain only one cell. The medium in each well is examined periodically. 1

4. Screening: The next step is a speedy primary screening system, in which the most effective hybridomas that produce antibodies of suitable specificity are identified. This screening is done by ELISA test. Antigen is applied in the bottom of the well of the culture plates. Samples of produced antibody is incubated in the wells. Antigen-antibody bind together. So, if the desired antibody is present in the sample then it’ll bind with the antigen and stay in the plates. While washing the bound materials are retained and the unbound materials are washed away. 1

5. Cloning: The B cell that produces the desired antibodies can be cloned to produce many same daughter clones. The cloning techniques are Limiting Dilution Method and Soft Agar Method. They can be used individually but most of the cases combined.
In limiting dilution method, 96-well plastic culture plate is used for cloning. The hybridoma cells are diluted and each cell is planted in each well. This process is repeated and it ensured that monoclonality is attained.
In the alternative method hybridoma cells are cloned in a single plate containing soft agar medium where the cells form spherical colonies. 1

6. Characterization and Storage: The monoclonality of the antibodies is established by biomedical and biophysical characterization of the antibodies using spectrometric, electrophoretic and chromatographic methods. The suitability and stability of the antibodies for therapeutic and diagnostic purpose is determined through this process.
Antibodies are preserved in frozen liquid nitrogen. Some are stored for running the further cloning process. 1

Kulkami, G. (2002). Chap-6, production of monoclonal antibody, Biotechnology and its applications in pharmacy, New Delhi: Jaypee Bros Medical Publishers.


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