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Hybridoma Technology - A Biotechnology TechniqueBY: Nidhi Uppangala | Category: Others | Submitted: 2010-08-31 10:05:41
Hybridoma technology is used to produce a hybrid cell. These hybrid cells are produced by fusing B-lymphocyte with tumour cell and they are called as myeloma cells. Thus these hybrid cells have got the ability to produce antibodies due to the B-lymphocyte genetic material and also capacity to divide indefinitely in the culture due to the presence of tumour cell or myeloma cells involved in the production of hybrid cells. Therefore, these hybrid cells produced from hybridoma technology are cultured in laboratory or passaged or subcultured using mouse peritoneal cavity and these cells produces monoclonal antibodies, and this technology is called as hybridoma technology.
Hybridoma technology was first discovered by G.Kohler and C.Milstein during 1975. They were also awarded Nobel Prize along with N.Jeme in Physiology and Medicine field during 1984. B-lymphocytes are pre-programmed to respond to a single type of antigen or antigenic determinant, therefore they produce single type of antibody specific to the specific antigen.
When an antigen reacts with B-lymphocyte receptors, lymphocytes divide rapidly and produce a clone of B cells, all these B cells produce antibodies against that specific antigen and this is called as clonal selection. That is B-lymphocytes produce only one type of antibodies which are specific to only one type of antigen or antigenic determinant. But fully differentiated antibody producing B-lymphocyte cells known as plasma cells does not divide when cultured in a laboratory.
Procedure of Hybridoma Technology:
1. B-lymphocytes are extracted from the spleen of an animal, but usually it is extracted from the mouse, which has been immunized with the required antigen against which monoclonal antibodies are produced. Mouse is immunized by giving antigen injection along with an adjuvant via subcutaneously or by peritoneal cavity; this is followed by booster doses of the antigen. Adjuvant is nonantigenic in nature but they stimulate the immune system.
2. This immunization with specific antigen increases the specific antibody producing B-lymphocytes; this considerably increases the chances of obtaining the required hybridoma cells or clones.
3. This specific antibody producing B-lymphocytes are then mixed with the selected myeloma cells and are induced to fuse to form hybrid cells. The myeloma cells are selected based on some criteria like these cells themselves should not produce antibodies and also they should contain a genetic markers such as HGPRT. This genetic marker helps in easy selection of the resulting hybrid cells.
4. When HGPRT myeloma cells are fused with specific antibody producing B-lymphocytes, the resulting cell population will have the mixture of cell population such as hybrid cells, myeloma cells, B-lymphocytes.
5. This mixture of cell population is then cultured in selective media known as HAT medium along with the drug aminopterin. The HGPRT myeloma cells cannot divide in the HAT medium due to the presence of aminopterin. The Specific antibody producing B-lymphocytes are unable to divide continuously in the culture medium, therefore eventually they die.
6. Only the hybridoma cells have got the ability to divide and proliferate on the HAT medium because genome from the B-lymphocyte makes them HGPRT positive and genome from the myeloma cells they can divide indefinitely. Thus only the hybridoma cells or fused cells are selected using selective media called as HAT medium.
Identification and Isolation of the Hybridoma Cells:
Hybridoma cells producing specific antibodies for the antigen used to immunize the animal (mouse) are identified and isolated by following method,
1. The specific antibodies present in the each microwell are identified using one of the methods such as precipitation method or agglutination method. Most commonly used and most sensitive and rapid method is ELISA (Enzyme Linked Immunosorbant Assay)
2. Wells which contain the antibodies specific to the antigens are identified and hybridoma cells are isolated from these wells and cultured (cloned). This ensures that these hybridoma cells have the capacity to produce same single type of antibodies specific to the antigen used.
3. After these hybridoma cells are multiplied using in vitro or in vivo method.
Mass Production of Antibodies:
4. The in vivo production involves injecting hybridoma cells into the peritoneal cavity of the animal (mouse), then ascetic fluid is isolated and then antibodies are isolated from it.
5. In vitro method hybridoma cells are cultured in suitable culture media and then antibodies are isolated and purified.
This hybridoma technology is used to produce monoclonal antibodies.
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