Hybridoma Technology For The Production Of Monoclonal Antibody Introduction
Hybridoma Technology For The Production Of Monoclonal Antibody
In 1970s the antibodies from the B cells are first identified. After that concept of hybridoma is created by Georges Kholer and Cesar Milstein by the fusion of the myeloma cells. Hybridoma cells are specialized cells which helps to produce large amount of antibodies. At last Greg Winter and his team were first produced monoclonal antibodies by using hybridoma technology. Monoclonal antibodies are derived from immune cells. They have monovalent affinity and they bind to the same epitope. 1
Uses of Monoclonal Antibody:
Monoclonal antibodies are used for varies important purpose. Such as,
-They are used for the analysis of parasite antigens
-They are used to differentiate B cells and T cells
-They are also used for identify different types of inflammatory cells .such as ‘ lymphocytes and leukocytes
-they are used to identify cancer antigens and anticancer agents to attack cancer metastases. 2
Production of hybridoma :
Hybridoma production has some important steps which helps to establish monoclonal antibodies
2. Cell Fusion
3. Selection page:1
5. Characterization and storage
Figure: Production Of Monoclonal Antibody By Using Hybridoma Technology.
At first, a mouse is injected by the mixture of an immunogen and an adjuvant. The injection is given mainly intravenously or subcutaneously .After that, the spleen of the mouse is collected and it is converted into solenocyte in the presence of suitable enzymes.
Cell fusion process is started by mixing the solenocytes and plasmacytoma cells. The mixing takes place in the 50% PEG medium. After some time it will produce hybridoma.
We know that, human body does not accept the antibody of another animal. As a result, they will produce harmful effects against then and the effectiveness will be decreased. Human monoclonal antibodies are produced by B cells and B cells are modified by using oncogenic virus. These antibodies can be used only after the elimination of such virus.
When the fusion is done the cells are transferred to a medium. The medium is known as HAT medium. After that the medium is incubated. Only hybridoma cells are workable and they are removed from the medium. After that, In 96 well plastic culture plates the liquates are transferred and they are tested for desired antibody reactivity.
ELISA is the most common medium for screening. The sample is placed in the well plates for incubation. The antibodies which has desire specificity will bound with the antigen and others will be washed of because they are unbound. There is tow page:3 components present there one that contains enzyme. In the screening the immunoconjugates are also retained. There will be second wash in which the colorless substance will be converted into colored substance by the enzyme the end of the screening the optical density of the product is measured with the ELISA readers.
Cloning means reproduction of the cells repeatedly. There are mainly two method such as, limiting dilution and soft agar. IN soft agar method malignant cells will proliferate in a semi solid medium. The process is continuously done until the monoclonal antibody is formed. Both methods are combined for cloning and the medium which is used for each screening is ELISA medium. Clones are cultured in the large vessel well and stocks are frozen for characterization.
7.Characterization And Storage:
In the characterization process the desire antibodies become monoclonal. The process is two types biochemical and biophysical.in the characterization process different characteristics of the antibodies are tested. They are, the suitability ,the stability, the affinity, the number of binding site, the capability of the antibodies to cope up with the frozen condition, the cell line is also very important to test.
At last, for storage a frozen condition is required for the stability of the monoclonal antibodies. For that purpose the stocks are stored in a liquid nitrogen in the different stages of the process. 3
1 Monoclonal antibody from Wikipedia from encyclopedia. Available from:
https://en.wikipedia.org/wiki/Monoclonal_antibodyaccessed 5 November 2018
2 Shivanand Pandey(2010)Hybridoma technology for production of monoclonal antibodies Smt. R. B. P. M. Pharmacy College, Atkot-360040, Rajkot, Gujarat. India
3 Kulkarni,G.(2002).Biotechnology and its applications in pharmacy.46th ed. New Delhi: Jaypee Bros Medical Publishers,pp.46-52.