From an exposure to antigens, antigenic molecules have several epitopes which result in synthesis of a mixture of antibodies. Each B-cell synthesizes a specific antibody. But antigens which contain multiple epitopes stimulate a series of B-cells which ultimately result in multiple cloning of plasma cells that are responsible for synthesizing different specific antibodies. This is termed as polyclonal activity. In 1975, Kohler and Milstein gave a concept of “one lymphocyte-one antibody” in the form of hybridoma. Each lymphocyte produces only one type of antibody for a specific antigenic epitopes and termed as monoclonal antibody. For unlimited supply of this monoclonal antibody, proper clone of B-cells and for harvesting pure monoclonal antibodies should be done by artificial means. This monoclonal antibody is produced by using a technology called “Hybridoma technology” which was discovered by George Kohler of West Germany and Cesal Milstein of Argentina in 1975.
In this technology, immortal hybridomas which are cell fusion of B-cells and myeloma cells are grown on artificial medium. These are further veiled for production of antibody specifically monoclonal antibodies for large scale. This technology for monoclonal antibody production opens a vast area of application specifically in medical sciences.
The fundamental of hybridoma technology is based on the immortalization of B lymphocyte cells having antibody producing capacity but limited growth characteristics in vitro. The lymphocytes are fused with cells from a non-antibody producing and myeloma (continuously growing tumor cell line), as the hybrids continue to secrete antibodies while gaining the immortality of the parent tumor cells. So monoclonal antibodies are ‘hybrid’ cells that are created by the fusion of two cells, which is a myeloma cell with genetic potential of multiplication characters, and another cell that has a gene that codes for the targeted antibody.
Methodology of hybridoma production
Production of hybridomas which secrete specific monoclonal antibodies needs several steps to complete. They are –
? Immunization of the test animals: Test animals are injected a specific immunogen mixed with an adjuvant at several sites of the body and at different times. After certain times the animal is assayed for specific antibodies for an optimum concentration. Then the animal is sacrificed for collecting the spleenocytes from the spleen.
Adapted from: Imgdealix.pw. (2018). The Immune System Hybridoma Technology Biochem t. online Available at: http://imgdealix.pw/The-Immune-System-Hybridoma-Technology-Biochem-t.html Accessed 4 Nov. 2018.
Figure: Production of monoclonal antibody by using hybridoma technology
? Cell fusion to produce hybridomas: The washed lymphocytes are mixed with myeloma cells in an appropriate medium which then exposed to PEG for a period of time and fusion takes place.
? Selection of the hybridomas: Hybridoma selection in HAT (Hypoxanthine, Aminopterine,Thymidine ) medium is followed by screening of the antibody specificity. After fusion is finished, the cells are transferred and incubated to HAT medium. When incubation is finished, the viable cells are our hybridomas and these cells are again transferred to the normal culture medium. The aliquots are distributed to 96-well plastic culture plates to test periodically for expected antibody reactivity.
? Screening of the antibodies: Hybridomas need to screened for desired antibody specificity. The cultures are tested for several times by using ELISA or RIA methods. These are done by binding to the antibody with specific antigens washed off to collect the desired cells for cloning desired antibodies.
Adapted from: Ib.bioninja.com.au. (2018). ELISA | BioNinja. online Available at: http://ib.bioninja.com.au/options/untitled/b4-medicine/elisa.html Accessed 4 Nov. 2018.
Figure: ELISA testing
? Cloning and propagation: The single cells that are secreting desired antibody are then propagated into the cell lines and cloned by using Limiting dilution method and Soft agar method.
• In the limiting dilution method, hybridoma cells are serially diluted and aliquoted into new wells as each well contains one cell only. This ensures that only monoclonal antibody is produced.
• In soft agar method, hybridoma cells are grown in a semisolid soft agar medium and form spherical colonies which will most likely to be monoclonal.
? Characterization and storage: For ensuring the monoclonality, we must check the biochemical and biophysical characters of the antibodies by spectrometric, electrophoretic and chromatographic methods. These antibodies are also tested for their suitability and intension of use like for therapeutic or diagnostic purposes.
The antibodies secreting from cell lines at several stages of cloning must be kept in the liquid nitrogen for further use and to skip several stages because it’s time consuming.
Application of Hybridoma technology
The purpose of hybridoma technique is the production of homogeneous antibody against a specific epitope of an antigen in vitro. These monoclonal antibodies have wide applications such as:
? Immunodiagnostic reagents, either for detection of the disease causing agents directly in tissues or body fluids or used in indirect diagnosis like serological detection of antibodies to the antigenic agent.
? Hybridoma technology is useful by producing broad spectrum of monoclonal antibody which are useful in cancer therapy, bone marrow transplantation, treating cardiovascular diseases like Myocardial infarction by Myoscint, Deep vein thrombosis by 99mTc-Fab fragment, Atherosclerosis etc.
? Hybridoma is now a hot cake technology for diagnosis of autoimmune diseases.
? For Biotechnological application the MoAbs is used for purification of protein, antigens, Drugs treating and Gene cloning ; expression.
? It is a research tool for the recognition of specific amino acid sequences of polypeptides.